Asthma associated factors as targets for treating atopic allergies including asthma and related disorders

ABSTRACT

A C to T DNA variation at position 3365 in exon 5 of the human Asthma Associated Factor 1 (AAF1) produces the predicted amino acid substitution of a methionine for a threonine at codon 117 of AAF1. When this substitution occurs in both alleles in one individual, it is associated with less evidence of atopic allergy including asthma, fewer abnormal skin test responses, and a lower serum total IgE. Thus, applicant has identified the existence of a non-asthmatic, non-atopic phenotype characterized by methionine at codon 117 when it occurs in both AAF1 gene products in one individual.

CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application is related to U.S. Provisional ApplicationSerial No. 60/002,765 which was filed Aug. 24, 1995.

FIELD OF THE INVENTION

[0002] This invention relates to regulating IL-9 activity and treatingatopic allergies and related disorders like asthma, based upon therelationship between IL-9 and its receptor.

BACKGROUND OF THE INVENTION

[0003] Inflammation is a complex process in which the body's defensesystem combats foreign entities. While the battle against foreignentities may be necessary for the body's survival, some defense systemsimproperly respond to foreign entities, even innocuous ones, asdangerous and thereby damage surrounding tissue in the ensuing battle.

[0004] Atopic allergy is an ecogenetic disorder, where geneticbackground dictates the response to environmental stimuli. The disorderis generally characterized by an increased ability of lymphocytes toproduce IgE antibodies in response to ubiquitous antigens. Activation ofthe immune system by these antigens leads to allergic inflammation andmay occur after ingestion, penetration through the skin, or afterinhalation. When this immune activation occurs and pulmonaryinflammation ensues this disorder is broadly characterized as asthma.Certain cells are critical to this inflammatory reaction and theyinclude T cells and antigen presenting cells, B cells that produce IgE,and mast cells/basophils and eosinophils that bind IgE. Theseinflammatory cells accumulate at the site of allergic inflammation andthe toxic products they release contribute to the tissue destructionrelated to the disorder.

[0005] While asthma is generally defined as an inflammatory disorder ofthe airways, clinical symptoms arise from intermittent air flowobstruction. It is a chronic disabling disorder that appears to beincreasing in prevalence and severity¹. It is estimated that 30-40% ofthe population suffer with atopic allergy, and 15% of children and 5% ofadults in the population suffer from asthma.¹ Thus, an enormous burdenis placed on our health care resources.

[0006] The mechanism of susceptibility to atopy and asthma remainsunknown. Interestingly, while most individuals experience similarenvironmental exposures, only certain individuals develop atopic allergyand asthma. This hypersensitivity to environmental allergens known as“atopy” is often indicated by elevated serum IgE levels or abnormallygreat skin test response to allergens in atopic individuals as comparedto nonatopics.¹⁰ Strong evidence for a close relationship between atopicallergy and asthma is derived from the fact that most asthmatics haveclinical and serologic evidence of atopy.⁴⁻⁹ In particular, youngerasthmatics have a high incidence of atopy.¹⁰ In addition, immunologicfactors associated with an increase in serum total IgE levels are veryclosely related to impaired pulmonary function.³

[0007] Both the diagnosis and treatment of these disorders areproblematic.¹ The assessment of inflamed lung tissue is often difficult,and frequently the source of the inflammation cannot be determined.Without knowledge of the source of the airway inflammation andprotection from the inciting foreign environmental agent or agents, theinflammatory process cannot be interrupted. It is now generally acceptedthat failure to control the pulmonary inflammation leads to significantloss of lung function over time.

[0008] Current treatments suffer their own set of disadvantages. Themain therapeutic agents, β agonists, reduce the symptoms, i.e.,transiently improve pulmonary functions, but do not affect theunderlying inflammation so that lung tissue remains in jeopardy. Inaddition, constant use of β agonists results in desensitization whichreduces their efficacy and safety.² The agents that can diminish theunderlying inflammation, the anti-inflammatory steroids, have their ownknown list of disadvantages that range from immunosuppression to boneloss.²

[0009] Because of the problems associated with conventional therapies,alternative treatment strategies have been evaluated.⁶⁵⁻⁶⁶ GlycophorinA,⁶⁴ cyclosporin,⁶⁵ and a nonapeptide fragment of IL-2,⁶³ all inhibitinterleukin-2 dependent T lymphocyte proliferation and therefore, IL-9production,⁵¹ however, they are known to have many other effects.² Forexample, cyclosporin is used as a immunosuppressant after organtransplantation. While these agents may represent alternatives tosteroids in the treatment of asthmatics,⁶³⁻⁶⁶ they inhibit interleukin-2dependent T lymphocyte proliferation and potentially critical immunefunctions associated with homeostasis. What is needed in the art is theidentification of a pathway critical to the development of asthma thatexplains the episodic nature of the disorder and the close associationwith allergy that is downstream of these critical immune functions.Nature demonstrated that this pathway is the appropriate target fortherapy since biologic variability normally exists at this pathway andthese individuals are otherwise generally not immunocompromised or illexcept for their symptoms of atopy.

[0010] Because of the difficulties related to the diagnosis andtreatment of asthma, the complex pathophysiology of this disorder isunder intensive study. Although this disorder is heterogeneous and maybe difficult to define because it can take many forms, certain featuresare found in common among asthmatics. Examples of such traits includeelevated serum IgE levels, abnormal skin test response to allergenchallenge, bronchial hyperresponsiveness [BHR], bronchodilatorreversibility, and airflow obstruction.³⁻¹⁰ These expressions of theseasthma related phenotypes may be studied as quantitative or qualitativemeasures.

[0011] Elevated IgE levels are also closely correlated with BHR, aheightened bronchoconstrictor response to a variety ofstimuli.^(4,6,8,9) BHR is believed to reflect the presence of airwayinflammation,^(6,8) and is considered a risk factor for asthma.¹¹⁻¹² BHRis accompanied by bronchial inflammation and an allergic diathesis inasthmatic individuals.¹³⁻²¹ Even in children with no symptoms of atopyand asthma, BHR is strongly associated with elevated IgE levels.¹⁹

[0012] A number of studies document a heritable component to atopy andasthma.^(4,10,21) However, family studies have been difficult tointerpret since these disorders are significantly influenced by age andgender, as well as many environmental factors such as allergens, viralinfections, and pollutants.²²⁻²⁴ Moreover, because there is no knownbiochemical defect associated with susceptibility to these disorders,the mutant genes and their abnormal gene products can only be recognizedby the anomalous phenotypes they produce. Thus, an important first stepin isolating and characterizing a heritable component is identifying thechromosomal locations of the genes.

[0013] Cookson et al. provided the first description of a geneticlocalization for inherited atopy.²⁵ These investigators describedevidence for genetic linkage between atopy and a single marker on aspecific chromosomal region designated 11q13.1. Later, they suggestedevidence of maternal inheritance for atopy at this locus.²⁶ Althoughmaternal inheritance [genetic imprinting] had been observed for atopy,it had never been explained previously. However, efforts to confirm thislinkage have not been generally successful.²⁷⁻³¹

[0014] Recently, the β subunit of the high-affinity IgE receptor wasmapped to chromosome 11q, and a putative mutation associated with atopyhas been described in this gene.^(32,33) However, because of thedifficulties by others of replicating this linkage, the significance ofthis gene and polymorphism remains unclear. While additional studieswill be required to confirm whether this putative mutation causes atopyin the general population, data collected so far suggests thispolymorphism is unlikely to represent a frequent cause of atopy.

[0015] Because serum IgE levels are so closely associated with the onsetand severity of allergy and asthma as clinical disorders, attention hasfocused on studies of the genetic regulation of serum total IgE levels.While past studies have provided evidence for Mendelian inheritance forserum total IgE levels,³⁴⁻³⁸ an indication of the existence of oneregulatory gene, others have found evidence for polygenic inheritance ofIgE, i.e., existence of several responsible genes.³⁹

[0016] Artisans have found several genes that may be important in theregulation of IgE and the development or progression of bronchialinflammation associated with asthma on chromosome 5q. They include genesencoding several interleukins, such as IL-3, IL-4, IL-5, IL-9, IL-13,granulocyte macrophage colony stimulating factor [GM CSF], a receptorfor macrophage colony stimulating factor [CSF-1R], fibroblast growthfactor acidic [FGFA], as well as others.⁴⁰ Recent evidence from familystudies suggests genetic linkage between serum IgE levels and DNAmarkers in the region of these candidate genes on chromosome 5q.^(41,42)Together, these investigations suggest that one or more major genes inthe vicinity of the interleukin complex on chromosome 5q regulates asignificant amount of the observed biologic variability in serum IgEthat is likely to be important in the development of atopy and asthma.

[0017] Linkage [sib-pair analyses] was also used previously to identifya genetic localization for BHR.⁷⁹ Because BHR was known to be associatedwith a major gene for atopy, chromosomal regions reported to beimportant in the regulation of serum IgE levels were examined.⁴²Candidate regions for atopy have been identified by linkage analyses.These studies identified the existence of a major gene for atopy onhuman chromosome 5q31-q33.⁴²

[0018] Therefore, to determine the chromosomal location of a gene[s]providing susceptibility to BHR, which would be coinherited with a majorgene for atopy, experiments were carried out using linkage analysesbetween BHR and genetic markers on chromosome 5q.^(42,79,82) Individualswith BHR were identified by responsiveness to histamine. Markers usefulfor mapping asthma-related genes are shown in FIG. 1.

[0019] Specifically, gene candidates for asthma, bronchialhyperresponsiveness, and atopy are shown [right] in their approximatelocation relative to the markers shown. The map includes the interleukingenes IL-4, IL-13, IL-5, and IL-3; CDC25, cell division cycle-25; CSF2,granulocyte-macrophage colony stimulating factor [GMCSF]; EGRL earlygrowth response gene-1; CD14, cell antigen 14; ADRB2, the β2-adrenergicreceptor; GRL1, lymphocyte-specific glucocorticoid receptor; PDGFR,platelet-derived growth factor receptor. Bands 5q31-q33 extendapproximately from IL-4 to D5S410. The distances reported aresex-averaged recombination fractions.

[0020] Affected sib-pair analyses demonstrated statistically significantevidence for linkage between BHR and D5S436, D5S658, and several othermarkers located nearby on chromosome 5q31-q33.⁷⁹ These data stronglysupported the hypothesis that one or more closely spaced gene[s] onchromosome 5q31-q33 determine susceptibility to BHR, atopy, andasthma.^(79,80,81,82)

[0021] Recently linkage has also been demonstrated between the asthmaphenotype and genetic markers on chromosome 5q31-q33.⁸³ This region ofthe human genome was evaluated for linkage with asthma because of thelarge number of genes representing reasonable positional candidates forproviding genetic susceptibility for atopy and BHR.

[0022] Linkage was demonstrated using the methods describedabove.^(42,83) Specifically, 84 families were analyzed from theNetherlands with both sib-pair and LODs for markers from this sameregion of chromosome 5q previously shown to be linked to BHR andatopy.^(42,83) An algorithm was used to categorize obstructive airwaysdisease in the asthmatic probands and their families. Thisclassification scheme was based, as described previously, on thepresence or absence of BHR to histamine, respiratory symptoms,significant smoking history [>5 pack years], atopy as defined by skintest response, airway obstruction [FEV1 % predicted <95% CI] andreversibility to a bronchodilator [>9% predicted].

[0023] Evidence was found for linkage between asthma and markers onchromosome 5q by affected sib pair analysis (N=10, P<0.05) and bymaximum likelihood analysis with a dominant model for the asthmaphenotype.⁸³

[0024] Asthma was linked to D5S658 with a maximal LOD of 3.64 at θ=0.03,using a dominant model [class 1 affected, class 2-4 uncertain, class 5unaffected] with a gene frequency of 0.015 [prevalence of 3%]. A maximalLOD of 2.71 at θ=0.0 was observed for D5S470 which is approximately 5 cMtelomeric, or away from IL-9, relative toD5S436.^(83 Subsequent to the original filing of this application, IL-)9or a gene nearby was suggested as likely to be important use atopy andasthma.⁴³ The IL-9 suggestion was based on a strong correlation in arandomly ascertained population between log serum total IgE levels andalleles of a genetic marker in the IL-9 gene.⁴³ This type of associationwith one or more specific alleles of a marker is termed “linkagedisequilibrium”, and generally suggests that a nearby gene determinesthe biologic variability under study.⁴⁴

[0025] The IL-9 gene has been mapped to the q31-q33 region of chromosome5.⁴⁰ Only a single copy of the gene is found in the humangenome.^(45,46) Structural similarity has been observed for the humanand murine IL-9 genes.^(45,46) Each gene consists of five exons and fourintrons extending across approximately four Kb of DNA. Expression of thegene appears to be restricted to activated T cells.^(45,46)

[0026] The functions of IL-9 now extend well beyond those originallyrecognized. While IL-9 serves as a T cell growth factor, this cytokineis also known to mediate the growth of erythroid progenitors, B cells,mast cells, and fetal thymocytes.^(45,46) IL-9 acts synergistically withIL-3 in causing mast cell activation and proliferation.⁴⁷ This cytokinealso potentiates the IL-4 induced production of IgE, IgG, and IgM bynormal human B lymphocytes.⁴⁸ IL-9 also potentiates the IL-4 inducedrelease of IgE and IgG1 by murine B lymphocytes.⁴⁹ A critical role forIL-9 in the mucosal inflammatory response to parasitic infection hasalso been demonstrated.^(50,51)

[0027] In addition to IL-9, chromosome 5q bears numerous other genecandidates including IL-3, IRF1, EGR1, ITK, GRL1, ADRB2, CSF1R, FGFA,ITGA2,CD14, PDGFR, CDC25, CSF2, IL-4, IL-5, IL-12B, and IL-13. These mayall be important in atopic allergy and as potential targets fortherapeutic development. Moreover, the art lacks any knowledge regardinghow the sequence of IL-9 or the function of IL-9 specifically correlateswith atopic allergy, asthma, or bronchial hyperresponsiveness. Withoutsuch knowledge, artisans would not know how or whether to use IL-9 toeither diagnose or treat these disorders.

[0028] The art does provide that IL-9 is a novel cytokine having anapparent molecular weight of approximately between 20 to 30 kD asdetermined by sodium dodecyl sulfate polyacrylamide gel electrophoresisunder reducing conditions. It is produced as a 144 amino acid protein,that is processed to a 126 amino acid glycoprotein. Yang et al.⁸⁵disclose that the DNA sequence encoding IL-9 comprises approximately 630nucleotides, with approximately 450 nucleotides in the proper readingframe for the protein.

[0029] It is also known in the art that multiple protein isoforms may begenerated from a single genetic locus by alternative splicing.Alternative splicing is an efficient mechanism by which multiple proteinisoforms may be generated from a single genetic locus. Alternativesplicing is used in terminally differentiated cells to reversibly modifyprotein expression without changing the genetic content of the cells.These protein isoforms are preferentially expressed in different tissuesor during different states of cell differentiation or activation.Protein isoforms may have different functions and Alms and White havecloned and expressed a naturally occurring splice variant of IL-4,formed by the omission of exon 2, thus called IL-4δ2.⁸⁶ It was observedthat IL-4δ2 inhibits T-cell proliferation induced by IL-4.

[0030] However, the art lacks any knowledge about IL-9 protein isoformswhich are formed-by deletions of exons 2 and 3 or the regulatoryfunctions exhibited by these truncated proteins. Specifically, theirrole in regulating the biological activity, namely, the down-regulationof IL-9 expression or activity is unclear. Moreover, the formation ofsuch isoforms by alternative splicing has not been previously observedor used to provide variants of IL-9 which function as agonists orantagonists of the native cytokine.

[0031] The art also lacks any knowledge about the role of the IL-9receptor with asthma-related disorders. It is known that IL-9 binds to aspecific receptor expressed on the surface of target cells.^(46,52,53)The receptor actually consists of two protein chains: one protein chain,known as the IL-9 receptor, binds specifically with IL-9 and the otherprotein chain is the chain, which is shared in common with the IL-2receptor.⁴⁶ In addition, the human IL-9 receptor cDNA has beencloned.^(46,52,53) This cDNA encodes a 522 amino acid protein whichexhibits significant homology to the murine IL-9 receptor. Theextracellular region of the receptor is highly conserved, with 67%homology existing between the murine and human proteins. The cytoplasmicregion of the receptor is less highly conserved. The human cytoplasmicdomain is much larger than the corresponding region of the murinereceptore.⁴⁶

[0032] The IL-9 receptor gene has also been characterized.⁵³ It isthought to exist as a single copy in the mouse genome and is composed ofnine exons and eight introns.⁵³ The human genome contains at least fourIL-9 receptor pseudogenes. The human IL-9 receptor gene has been mappedto the 320 kb subtelomeric region of the sex chromosomes X and Y.⁴⁶Nonetheless, despite these studies, the art lacks any knowledge of arelation between the IL-9 receptor and atopic allergy, asthma, orbronchial hyperresponsiveness.

[0033] Thus, the art lacks any knowledge of how the IL-9 gene, itsreceptor, and their functions, are related to atopic allergy, asthma,bronchial hyperresponsiveness, and related disorders. Therefore, thereis a specific need in the art for genetic information on atopic allergy,asthma, bronchial hyperresponsiveness, and for elucidation of the roleof IL-9 in the etiology of these disorders. There is also a need forelucidation of the role of the IL-9 receptor and the IL-9 receptor genein these disorders. Furthermore, most significantly, based on thisknowledge, there is a need for the identification of agents which arecapable of regulating the interaction between IL-9 and its receptor fortreating these disorders.

SUMMARY OF THE INVENTION

[0034] Applicant has satisfied the long felt need for a treatment foratopic allergy including asthma and related disorders by providinginformation demonstrating the role of IL-9 (also known as AsthmaAssociated Factor 1, or AAFI) in the pathogenesis of these disorderswhich information has led to compounds that are capable of regulatingthe activity of IL-9. Applicant has also demonstrated conserved linkageand synteny homologies between mice and humans for a gene thatdetermines biologic variability in airway hyperresponsiveness. Theserelationships specifically identify IL-9 as a gene candidate. Inaddition, applicant has determined that IL-9 is critical to a number ofantigen-induced responses in mice including bronchialhyperresponsiveness, eosinophilia and elevated cell counts in bronchiallavage, and elevated serum total IgE. These findings typify the allergicinflammation associated with asthma.

[0035] Furthermore, applicant has determined that a C to T nucleic acidvariation at position 3365 in exon 5 of the human IL-9 gene produces thepredicted amino acid substitution of a methionine for a threonine atcodon 117 of IL-9. When this substitution occurs in both alleles in oneindividual, it is associated with less evidence of atopic allergyincluding asthma, fewer abnormal skin test responses, and a lower serumtotal IgE. Thus, applicant has identified the existence of anonasthmatic, nonatopic phenotype characterized by methionine at codon117 when it occurs in both IL-9 gene products in one individual. As anadditional significant corollary, applicant has identified the existenceof susceptibility to an asthmatic, atopic phenotype characterized by athreonine at codon 117. Thus, the invention includes purified andisolated DNA molecules having such a sequence as well as the peptidesencoded by such DNA.

[0036] The biological activity of IL-9 results from its binding to theIL-9 receptor and the consequent propagation of a regulatory signal inspecific cells. Therefore, IL-9 functions can be interrupted orregulated by the interaction of IL-9 agonists or antagonists with IL-9or its receptor. Down regulation, i.e. reduction of the functionscontrolled by IL-9, is achieved in a number of ways. Administeringagonists or antagonists that can interrupt the binding of IL-9 to itsreceptor is one key mechanism and such agonists and antagonists arewithin the claimed invention. Examples include administration ofpolypeptide products encoded by the DNA sequences of IL-9 or IL-9receptor wherein the DNA sequences contain various mutations. Thesemutations may be point mutations, insertions, deletions, orspliced-variants of IL-9 or its receptor.

[0037] A further embodiment of this invention includes the regulation ofthe activity of IL-9 by administering “agonists and antagonists.” Theskilled artisan will readily recognize that all molecules containing therequisite 3-dimensional structural conformation and which contain theresidues essential or critical for receptor binding are within the scopeof this invention. Specifically, residues 43-60 and 71-90 of the matureprotein appear to be important for receptor binding. Applicant has shownthat peptides KP-16 (residues 43-60) and KP-20 (residues 71-90) act asreceptor antagonists. In addition, these residues in the native IL-9molecule are predicted to form anti-parallel helical structures. Thethree dimensional structure of the protein suggests that specificallyserine 52 and/or glutamic acid 53 interact with lysine 85, serine 56interacts with lysine 82, and threonine 59 interacts with valine 78. Thethree dimensional coordinates of these anti-parallel helices and therelated functional groups represent the requisite 3-dimensionalconformation critical for receptor binding and compounds which simulatethese relationships are within the scope of this invention.

[0038] The biological activity of the IL-9 receptor (also called AsthmaAssociates Factor 2, AAF2) can also be modulated by using soluble IL-9receptor molecules. Such a molecule prevents the binding of IL-9 to thecell-bound receptor and acts as an antagonist for IL-9, and is alsowithin the scope of this invention.

[0039] Polyclonal and monoclonal antibodies which block the binding ofIL-9 to its receptor are also within the scope of this invention and areuseful therapeutic agents in treating atopic allergy including asthmaand related disorders.

[0040] Another embodiment of this invention relates to the use ofisolated DNA sequences containing various mutations such as pointmutations, insertions, deletions, or spliced mutations of IL-9 or theIL-9 receptor in gene therapy.

[0041] Expression of IL-9 and IL-9 receptor is also down-regulated byadministering an effective amount of synthetic antisense oligonucleotidesequences. The oligonucleotide compounds of the invention bind to themRNA coding for human IL-9 and IL-9 receptor thereby inhibitingexpression of these molecules.

[0042] The structure of both IL-9 and the IL-9 receptor have beenexamined and analyzed in great detail and amino acid residues of IL-9critical for receptor binding have been identified. Based on structuralstudies and the binding characteristics of this specific binding pair,this invention further includes small molecules tailored such that theirstructural conformation provides the residues essential for blocking theinteraction of IL-9 with the IL-9 receptor. Such blockade results inmodulation of the activity of the receptor and these molecules are,therefore, useful in treating atopic allergies.

[0043] Another embodiment of this invention is directed to theregulation of downstream signaling pathways necessary for IL-9 function.IL-9 induces tyrosine phosphorylation of Stat3 which appears to beunique to the IL-9 signaling pathway⁵⁸ and is useful as a target forinhibitors. Specific and nonspecific inhibitors of tyrosine kinase suchas tyrophostins are, therefore, useful in downstream regulation of thephysiological activity of IL-9, and are part of the invention.

[0044] In a further embodiment aminosterol compounds are also useful intreating atopic allergies and related disorders because they are alsoinvolved in blocking signal transduction of the IL-9 signal transductionpathway.

[0045] The products discussed above represent various effectivetherapeutic agents in treating atopic allergies, asthma and otherrelated disorders.

[0046] This invention also includes the truncated polypeptides encodedby the DNA molecules described above. These polypeptides are capable ofregulating the interaction of IL-9 with the IL-9 receptor.

[0047] Thus, applicant has identified the critical role of the IL-9pathway in pathogenesis of atopic allergy, including bronchialhyperresponsiveness, asthma, and related disorders. More specifically,applicant has provided antagonists and methods of identifyingantagonists that are capable of regulating the interaction between IL-9and its receptor. Applicant also provides methods for regulating theactivity of IL-9 by: 1) administering a compound having activitycomparable to IL-9 containing methionine at codon 117 and the ability tobind to a receptor for IL-9 in an amount sufficient to down-regulate theactivity of IL-9; and 2) by administering truncated protein productsencoded by isolated nucleic acid sequences comprising deletions of anyone or more of exons 1, 2, 3, 4, or 5.

[0048] Having identified the critical role of the IL-9 pathway in atopicallergy, bronchial hyperresponsiveness, and asthma, applicant alsoprovides a method for the diagnosis of susceptibility to atopic allergy,asthma, and related disorders. Lastly, applicant provides a method forassaying the functions of IL-9 and its receptor to identify compounds oragents that may be administered in an amount sufficient to down-regulateeither the expression or functions of IL-9 and the IL-9 receptor.

[0049] The accompanying figures, which are incorporated in andconstitute a part of this specification, illustrate several embodimentsof the invention and, together with the description, serve to explainthe principle of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

[0050]FIG. 1: Map showing the relative order and distance incentiMorgans [cM] between the polymorphic genetic markers useful formapping asthma-related genes.

[0051]FIG. 2: Illustration of the genetic map of human chromosome5q31-q33 and syntenic regions in the mouse.

[0052]FIG. 3: The LOD score curve on mouse chromosome 13 foratracurium-induced airway responsiveness in mice with increasedsusceptibility to bronchoconstrictor stimuli.

[0053]FIG. 4: Alignment of amino acid sequences corresponding to exon 5of the human and murine IL-9 genes. The first sequence is translatedfrom the Thr allele of the human gene. The middle sequence is translatedfrom the Met allele of the human gene. The final sequence is translatedfrom the murine gene.

[0054]FIG. 5: Histogram of the correlation between human IL-9 genealleles and serum total IgE titers measured in international units. S/Sdenotes Thr/Thr individuals, S/R denotes Thr/Met individuals and R/Rdenotes Met/Met individuals.

[0055]FIG. 6: Illustration the simple sequence repeat polymorphism atthe IL-9 locus.

[0056]FIG. 7: Translated cDNA sequence of Thr117 version of IL-9.

[0057]FIG. 8: Translated cDNA sequence of Met117 version of IL-9.

[0058]FIG. 9: Map of pFlag expression construct with Thr117.

[0059]FIG. 10: Sequence of pFlag expression construct for the Thr117version of the cDNA from the region surrounding the site of ligation.

[0060]FIG. 11: Map of pFlag expression construct with Met117.

[0061]FIG. 12: Sequence of pFlag expression construct for the Met117version of the cDNA from the region surrounding the site of ligation.

[0062]FIG. 13: Western blot of recombinant IL-9 proteins

[0063]FIG. 14: Amino acid sequences for inhibitory peptides.

[0064]FIG. 15: Inhibition by KP-16 of IL-9 mediated MO7e proliferation.

[0065]FIG. 16: Inhibition by KP-20 of IL-9 mediated MO7e proliferation.

[0066]FIG. 17: Inhibition by KP-23 of IL-9 mediated MO7e proliferation.

[0067]FIG. 18: Inhibition by various tyrophostins of IL-9 mediated MO7eproliferation.

[0068]FIG. 19: Inhibition by various aminosterols of IL-9 mediated MO7eproliferation.

[0069]FIG. 20: Characterization of the role of IL-9 in the antigenresponse in vivo.

[0070]FIG. 21: Histologic examination of lungs from control, ovachallenged, and anti-IL-9 pretreated animals.

[0071]FIG. 22: Inhibition of the antigen response in vivo by blockingantibodies to the murine IL-9 receptor.

[0072]FIG. 24: Expression of human Met117 IL-9 and Thr117 IL-9.

[0073]FIG. 25: Binding of the human recombinant Met117 and Thr117 formsof IL-9 to a soluble receptor.

[0074]FIG. 26: Steady state levels of IL-9 in unstimulated andstimulated murine splenocytes.

[0075]FIG. 27: An appendix of chemical moieties.

[0076]FIG. 28: Aminosterols isolated from the dog fish shark.

DETAILED DESCRIPTION OF THE INVENTION

[0077] Applicant has resolved the needs in the art by elucidating anIL-9 pathway and compositions that affect that pathway that may be usedin the diagnosis, prevention or treatment of atopic allergy includingasthma and related disorders. Asthma encompasses inflammatory disordersof the airways with reversible airflow obstruction. Atopic allergyrefers to atopy, and related disorders including asthma, bronchialhyperresponsiveness (BHR), rhinitis, urticaria, allergic inflammatorydisorders of the bowel, and various forms of eczema. Atopy is ahypersensitivity to environmental allergens expressed as the elevationof serum total IgE or abnormal skin test responses to allergens ascompared to controls. BHR refers to bronchial hyperresponsiveness, aheightened bronchoconstrictor response to a variety of stimuli.

[0078] By analyzing the DNA of families that exhibit asthma-relateddisorders, applicant has identified a polymorphism in the IL-9 gene thatcorrelates with the biologic variability of serum total IgE as onemeasurable expression of atopy. The IL-9 gene (also known as AsthmaAssociated Factor 1 or AAF1) refers to the genetic locus ofinterleukin-9, a cytokine exhibiting a variety of functions involvingthe regulation of human myeloid and lymphoid systems. The IL-9 gene ofthe present invention is found in the q31-q33 region of human chromosome5 and chromosome 13 in the mouse.

[0079] By polymorphism, applicant means a change in a specific DNAsequence, termed a “locus”, from the prevailing sequence. In general, alocus is defined as polymorphic when artisans have identified two ormore alleles encompassing that locus and the least common allele existsat a frequency of 1% or more.

[0080] The polymorphism of the present invention leads to an amino acidsubstitution at residue 117 of IL-9. Specifically, instead of thehydrophilic amino acid threonine, the IL-9 of the present inventionexhibits the hydrophobic amino acid methionine (Met IL-9). On a geneticlevel, the polymorphism of the present invention is a substitution of athymine residue for a cytosine residue at nucleotide position 3365 inthe human IL-9 gene as it is described by Renauld and colleagues(1990)[GenBank accession numbers M30135 and M30136],⁵⁴ or at the comparablenucleotide position 4244 of the human IL-9 gene sequence reported byKelleher et al.,(1991) [GenBank accession number M86593].⁵⁵

[0081] Individuals with a threonine (Thr) at amino acid 117 of IL-9 ineither one or both of their alleles (Thr/Thr or Thr/Met) generallyexhibit susceptibility to an asthmatic or atopic allergic phenotype, andthese genotypes are characterized by higher mean serum total IgE levelsin the populations studied. In contrast, those individuals with amethionine (Met) at codon 117 of IL-9 in both alleles (Met/Met) exhibita lack of asthma, fewer abnormal skin test responses, and a lower serumtotal IgE. Thus, the Met/Met genotype of IL-9 appears to protect againstasthma or atopic allergy.

[0082] Accordingly, the invention provides a purified and isolated DNAmolecule comprising a nucleotide sequence encoding human interleukin 9containing methionine at position 117 (Met IL-9), or a fragment thereof.The invention also includes degenerate sequences of the DNA as well assequences that are substantially homologous. The source of the IL-9 ofthe invention is human. Alternatively, the DNA or fragment thereof maybe synthesized using methods known in the art. It is also possible toproduce the compound by genetic engineering techniques, by constructingDNA by any accepted technique, cloning the DNA in an expression vehicleand transfecting the vehicle into a cell which will express thecompound. See, for example, the methods set forth in Sambrook et al.,MOLECULAR CLONING: A LABORATORY MANUAL, 2d ed. Cold Spring HarborLaboratory Press [1985].

[0083] Airway hyperresponsiveness is found in virtually all asthmaticsand in some strains of inbred mice (DBA/2).⁸⁴ Airway hyperresponsivenessis a risk factor for the development of asthma in humans and is used inanimal models of asthma as a physiologic measure to assess the efficacyof treatment for asthma. These data along with human⁷⁹ and murinegenetic mapping results (see Examples 1 and 2) suggest a critical rolefor the murine IL-9 gene product in the airway response of the mouse. Inparticular, the hyperresponsive DBA/2(D2) mice differ from theC57BL/6(B6)hyporesponsive mice⁸⁴ in their expression of steady statelevels of IL-9 (See Example 14, FIG. 26). Furthermore, pretreatment withblocking antibodies to IL-9/IL-9 receptor can optionally providecomplete protection from antigen induced airway hyperresponsiveness andinflammation in mice demonstrating a critical regulatory role for IL-9in these immune responses. Thus, these data demonstrate that althoughdifferent molecular changes produce biologic variability in airwayresponsiveness in humans and mice, these changes arise in the samegene(s) (IL-9/IL-9R) that regulate this pathway. Taken together, theseobservations confirm the critical role of IL-9 and the IL-9 receptor inairway hyperresponsiveness, asthma, and atopic allergy. Moreover, thesedata demonstrate that agents of the convention, which block theinteraction of IL-9 with its receptor, protect against an antigeninduced response such as those detailed above.

[0084] Further evidence defining the critical role of IL-9 in thepathogenesis of atopic allergy, bronchial hyperresponsivenss, asthma,and related disorders derives directly from the applicants observationthat IL-9 is critical to a number of antigen induced responses in mice.When the functions of IL-9 are down regulated by antibody pretreatmentprior to aerosol challenge with antigen, the animals can be completelyprotected from the antigen induced responses. These responses include:bronchial hyperresponsiveness, eosinophilia and elevated cell counts inbronchial lavage, histologic changes in lung associated withinflammation, and elevated serum total IgE. Thus, the treatment of suchresponses, which are critical to the pathogenesis of atopic allergy andwhich characterize the allergic inflammation associated with asthma, bythe down regulation of the functions of IL-9, are within the scope ofthis invention.

[0085] Applicant also teaches the regulation of the activity of IL-9 byadministering “agonists and antagonists” to the IL-9 receptor. Theskilled artisan will readily recognize that all molecules containing therequisite 3-dimensional structural conformation and which contain theresidues essential or critical for receptor binding are within the scopeof this invention. Applicant has shown that peptides KP-16 (IL-9residues 43-60) and KP-20 (IL-9 residues 71-90) (produced using standardpeptide automated synthesis techniques, for example, the AppliedBiosystems Model 431A Peptide Synthesizer) act as IL-9 antagonists.Specifically, applicant demonstrates that residues 43-60 and 71-90 ofthe mature protein appear to be important for receptor binding. Inaddition, these residues include most of exon 4 (amino acids 44-88) andare predicted to form anti-parallel helical structures. The threedimensional structure of the protein suggests that specifically serine52 and/or glutamic acid 53 interact with lysine 85, serine 56 interactswith lysine 82, and threonine 59 interacts with valine 78. The threedimensional coordinates of these parallel helices and the relatedfunctional groups represent the requisite 3-dimensional conformationcritical for receptor binding and compounds that simulate theserelationships are within the scope of this invention.

[0086] The demonstration of an IL-9 sequence associated with anasthma-like phenotype, and one associated with the lack of anasthma-like phenotype, indicates that the lungs' inflammatory responseto antigen is dependent on IL-9, and therefore, that down regulating thefunction of IL-9 should protect against the antigen induced response.Furthermore, applicant also provides methods of diagnosingsusceptibility to atopic allergy and related disorders and for treatingthese disorders based on the relationship between IL-9 and its receptor.

[0087] A receptor is a soluble or membrane bound component thatrecognizes and binds to molecules, and the IL-9 receptor (also known asAsthma Associated Factor 2 or AAF2) of the invention is the componentthat recognizes and binds to IL-9. The functions of the IL-9 receptorconsist of binding an IL-9-like molecule and propagating its regulatorysignal in specific cells.⁵⁷⁻⁶⁰ An interruption of that function willlead to a down regulation, i.e., reduction, of either the expression ofIL-9 or of the functions controlled by IL-9. Accordingly, by virtue ofthis interaction between. IL-9 and the IL-9 receptor, certain functionsof the organism are modulated or controlled. For a general discussion ofreceptors, see Goodman and Gilman's The Pharmacologic Basis ofTherapeutics (Seventh Edition, MacMillan Publishing Company, N.Y. USA,1985).

[0088] One diagnostic embodiment involves the recognition of variationsin the DNA sequence of IL-9. One method involves the introduction of anucleic acid molecule (also known as a probe) having a sequencecomplementary to the IL-9 of the invention under sufficient hybridizingconditions, as would be understood by those in the art. In oneembodiment, the sequence will bind specifically to the Met117 IL-9 or toThr117 IL-9, and in another embodiment will bind to both Met117 IL-9 andThr117 IL-9. Another method of recognizing DNA sequence variationassociated with these disorders is direct DNA sequence analysis bymultiple methods well known in the art.⁷⁷ Another embodiment involvesthe detection of DNA sequence variation in the IL-9 gene associated withthese disorders.⁷³⁻⁷⁷ These include the polymerase chain reaction,restriction fragment length polymorphism (RFLP) analysis and singlestranded conformational analysis. In a preferred embodiment, applicantprovides specifically for a method to recognize, on a genetic level, thepolymorphism in IL-9 associated with the Thr and Met alleles using aStyI RFLP as described herein. In other embodiments Nla, Pfim1, PflM1,and Nco1 RFLPs may be used to distinguish these two alleles of IL-9genes.

[0089] Another embodiment involves treatment of atopic allergy andrelated disorders. In a preferred embodiment, the applicant provides amethod of administering a compound having activity comparable to MetIL-9 and the ability to bind to an IL-9 receptor in an amount sufficientto down regulate the activity of IL-9. A compound having activitycomparable to Met IL-9 is a compound that functions similarly but notnecessarily identically. Thus, it may bind to the IL-9 receptor butwithout the same physiological effects. Examples include amino acidsequences of IL-9 containing various point mutations and/or deletionsand sequences substantially homologous thereto. For example, such acompound may interrupt the binding of Thr IL-9 to the IL-9 receptor asmeasured by techniques known in the art. The invention also encompassesfunctionally effective fragments of the above amino acid sequences. Inone such technique, the Thr IL-9 may be considered a “ligand” for theIL-9 receptor, and binding between the two may be assessed byligand-binding assays which are well known in the art as set forth inGoodman and Gilman's The Pharmacologic Basis of Therapeutics (SeventhEdition, MacMillan Publishing Company, N.Y. USA, 1985).

[0090] In another embodiment, the compound may resemble the Met alleleof IL-9 in structure. Thus, such a compound may incorporate a methioninein codon 117 of IL-9 or may incorporate another hydrophobic amino acid.Thus, included within the scope of this invention are IL-9 varientscomprising substitutions of Thr at position 117 by amino acids selectedfrom the group consisting of alanine, valine, leucine, isoleucine,proline, phenylalanine, tryptophan, and methionine. Alternatively, thecompound of the invention may exist as a fragment of IL-9 with astructural composition similar to Met IL-9. In another embodiment of theinvention, the compound may retain functions comparable to Met IL-9, butmay not resemble Met IL-9 in structure. For example, the composition ofthe compound may include molecules other than amino acids. This exampleis merely illustrative and one of ordinary skill in the art wouldreadily recognize that other substitutions and/or deletion analogs ofIL-9 resulting in effective antagonists are also within the scope ofthis invention. As discussed above all molecules containing therequisite 3-dimensional structural conformation and which contain theresidues essential or critical for receptor binding are within the scopeof this invention.

[0091] Specific assays may be based on IL-9's known regulation, in part,of the proliferation of T lymphocytes, IgE synthesis, and release frommast cells.⁵⁴⁻⁶⁰ Another assay involves the ability of human IL-9 tospecifically induce the rapid and transient tyrosine phosphorylation ofmultiple proteins in MO7e cells.⁵⁷ Because this response is dependent onthe expression and activation of the IL-9 receptor, it represents asimple method or assay for the characterization of potentially valuablecompounds. The tyrosine phosphorylation of Stat3 transcriptional factorappears to be specifically related to the actions of IL-9,⁵⁸ and thisresponse represents a simple method or assay for the characterization ofcompounds within the invention. Still another method to characterize thefunction of IL-9 and IL-9-like molecules involves the well known murineTS¹ clone and the D10 clone available from ATCC used to assess humanIL-9 function with a cellular proliferation assay.⁵⁹ The Met IL-9 thatforms a part of the invention may be viewed as a “weak agonist” of theIL-9 receptor. Such weak agonists are another preferred embodiment ofthe invention. The term agonist, according to this invention, includescompounds that mimic at least some of the effects of endogenouscompounds by interacting or binding with a receptor. Agonists thatinteract or bind to the IL-9 receptor on the surface of certain cellsinitiate a series of biochemical and physiological changes that arecharacteristic of this cytokine's actions.^(45-51,54-60) To identifyother weak agonists of the invention, one may test for binding to theIL-9 receptor or for IL-9-like functions as described herein and in thecited literature.^(2,45-51,54-60)

[0092] The present invention also includes antagonists of IL-9 and itsreceptor. Antagonists are compounds that are themselves devoid ofpharmacological activity but cause effects by preventing the action ofan agonist. To identify an antagonist of the invention, one may test forcompetitive binding with a known agonist or for down-regulation ofIL-9-like functions as described herein and in the citedliterature.^(2,45-51,54-60)

[0093] The binding of either the agonist or antagonist may involve allknown types of interactions including ionic forces, hydrogen bonding,hydrophobic interactions, van der Waals forces, and covalent bonds. Inmany cases, bonds of multiple types are important in the interaction ofan agonist or antagonist with a receptor.

[0094] In a further embodiment, these compounds may be analogs of IL-9.IL-9 analogs may be produced by point mutations in the isolated DNAsequence for the gene, nucleotide substitutions, and/or deletions whichcan be created by methods that are all well described in the art.⁶² Thisinvention also includes spliced variants of IL-9 and discloses isolatednucleic acid sequences of interleukin-9, which contain deletions of oneor more of its five exons. The term “spliced variants” as used hereindenotes a purified and isolated DNA molecule encoding human IL-9comprising at least one exon. There is no evidence of naturallyexpressed spliced mutants in the art. Thus, the present inventionprovides an isolated nucleic acid containing exons 1, 4 and 5 of humanIL-9. Other variants within the scope of this invention includesequences comprising exons 1, 2, 3, 4, and 5; exons 1, 2, 3, and 4;exons 1, 2, 4, and 5 and exons 1, 3, 4, and 5. It must be understoodthat these exons may contain various point mutations.

[0095] Specific examples of antagonistic peptides derived from IL-9include KP-16 (SEQ. ID No. 13) and KP-20 (SEQ. ID NO. 14) which arederived from exon 4. Exon 4 encodes 44 amino acids while the peptidesmentioned above contain 16 and 20 amino acids respectively and they donot overlap. These peptides exhibit considerable inhibitory activityboth individually and when assayed in combination. Additionally, KP-23(SEQ ID NO. 15) and KP-24 (SEQ ID NO 16) are derived from exon 5 andalso exhibit similar activity. Splice variants of IL-9 can be formed bydeletion of any one or more of the IL-9 exons 1 through 5. As shownabove, peptides derived from these exons show regulatory capability and,accordingly, are useful in treating atopic allergies, including asthma.

[0096] It is known in the art that, in multienzyme systems, the first orregulatory enzyme can be activated or inhibited by the end product ofthe multi-enzyme system. When the concentration of the end productincreases over the steady state concentration, the end product will actas a specific activator or inhibitor of the regulatory enzyme in thesequence. Such feedback mechanism is also relevant to the IL-9 systemand it is observed that the various polypeptides of this invention arecapable of exerting such activation or inhibitory control on theactivity of the IL-9 receptor and possibly the expression or function ofother cytokines and their receptors that play a role in the pathogenesisof asthma.

[0097] The invention also includes modifications of agonists orantagonists that can be made using knowledge that is routine to those inthis art. For example, the affinity of a compound for a receptor isgenerally closely related to the chemical structure of the compound.Thus, structure-activity relationships may be used to modify theagonists and antagonists of the invention. For example, the techniquesof crystallography/X-ray diffraction and NMR may be used to makemodifications of the invention.

[0098] For example, one can create a three dimensional structure ofhuman IL-9 that can be used as a template for building structural modelsof deletion mutants using molecular graphics. These models can then beused to identify and construct a mutant IL-9 molecule with affinity forthe IL-9 receptor comparable to IL-9, but with a lower biologicactivity. What is meant by lower biologic activity is 2 to 100,000 foldless than IL-9, preferably 100 to 1,000 fold less than IL-9.

[0099] In still another embodiment, these compounds also may be used asdynamic probes for receptor structure and to develop receptorantagonists using IL-9 dependent cell lines.

[0100] In addition, this invention also provides compounds that preventthe synthesis or reduce the biologic stability of IL-9 or the IL-9receptor. Biologic stability is a measure of the time between thesynthesis of the molecule and its degradation. For example, thestability of a protein, peptide or peptide mimetic⁸⁹ therapeutic may beprolonged by using D-amino acids, or shortened by altering its sequenceto make it more susceptible to enzymatic degradation.

[0101] In another embodiment, the agonists and antagonists of theinvention are antibodies to IL-9 and the IL-9 receptor. The antibodiesto IL-9 and its receptor may be either monoclonal or polyclonal madeusing standard techniques well known in the art (See Harlow & Lane'sAntibodies—A Laboratory Manual (Cold Spring Harbor Laboratory, 1988).They can be used to block IL-9 from binding to the receptor. In oneembodiment the antibodies interact with IL-9. In another embodiment theantibodies interact with the IL-9 receptor. The IL-9 used to elicitthese antibodies can be any of the IL-9 varients discussed above.Antibodies are also produced from peptide sequences of IL-9 or the IL-9receptor using standard techniques in the art (see Protocols inImmunology, Chapt. 9, Wiley). The peptide sequences from the murine IL-9receptor that can be used to produce blocking antisera have beenidentified as: GGQKAGAFTC (residues 1-10)(SEQ ID NO:19);LSNSIYRIDCHWSAPELGQESR (residues 11-32)(SEQ ID NO:20); andCESYEDKTEGEYYKSHWSEWS (residues 184-203 with a Cys residue added to theN-terminus for coupling the peptide to the carrier protein)(SEQ IDNO:21). In addition, an epitope that binds to a blocking antibodydirected to the human IL-9 receptor has been identified as residues 8-14of the mature human IL-9 receptor. (TCLTNNI)(SEQ ID NO:22) and twoepitopes that bind to blocking antibodies directed to human IL-9 havealso been identified as residues 50-67 (CFSERLSQMTNTTMQTRY) (SEQ IDNO:23) and residues 99-116 (TAGNALTFLKSLLEIFQK) (SEQ ID NO:16) The humanepitopes as well as the human peptides that correspond to the peptidesthat produce blocking antibodies in the murine sequences are most likelyto be useful for the production of therapeutic antibodies.

[0102] In still another embodiment, the compounds of the invention maybe coupled to chemical moieties, including proteins that alter thefunctions or regulation of the IL-9 pathway for therapeutic benefit inatopic allergy and asthma.⁶¹ These proteins may include in combinationother cytokines and growth factors including⁶⁷ L-4, IL-5, IL-3, IL-2,IL-13, and IL-10 that may offer additional therapeutic benefit in atopicallergy and asthma. In addition, the IL-9 of the invention may also beconjugated through phosphorylation and conjugated to biotinylate,thioate, acetylate, iodinate, and any of the crosslinking reagents shownin FIG. 27 (Pierce).

[0103] In a further embodiment, the invention includes the downregulation of IL-9 expression or function by administering soluble IL-9receptor molecules that bind IL-9. Renauld et al.⁵⁹ have shown theexistence of a soluble form of the IL-9 receptor. This molecule can beused to prevent the binding of IL-9 to cell bound receptor and act as anantagonist of IL-9. Soluble receptors have been used to bind cytokinesor other ligands to regulate their function.⁸⁷ A soluble receptor is aform of a membrane bound receptor that occurs in solution, or outside ofthe membrane. Soluble receptors may occur because the segment of themolecule which commonly associates with the membrane is absent. Thissegment is commonly referred to in the art as the transmembrane domainof the gene, or membrane binding segment of the protein. Thus, in oneembodiment of the invention, a soluble receptor may represent a fragmentor an analog of a membrane bound receptor. In another embodiment of theinvention, the structure of the segment that associates with themembrane may be modified (e.g. DNA sequence polymorphism or mutation inthe gene) so the receptor is not inserted into the membrane, or thereceptor is inserted, but is not retained within the membrane. Thus, asoluble receptor, in contrast to the corresponding membrane bound form,differs in one or more segments of the gene or receptor protein that areimportant to its association with the membrane.^(52,53)

[0104] These compounds may be known forms of a soluble IL-9 receptorthat act to bind IL-9. Alternatively, these compounds may resemble knownforms of the IL-9 receptor, but may exist as fragments. In anotherembodiment of the invention, the compound may retain functionscomparable to soluble IL-9 receptor, but may not resemble soluble IL-9receptor in composition. For example, the composition of the compoundmay include molecules other than amino acids. Thus, these compounds willbind IL-9 and prevent IL-9 from acting at its cell surface receptor.

[0105] A further embodiment of the invention relates to antisense orgene therapy. It is now known in the art that altered DNA molecules canbe tailored to provide a specific selected effect, when provided asantisense or gene therapy. The native DNA segment coding for IL-9receptor, has, as do all other mammalian DNA strands, two strands; asense strand and an antisense strand held together by hydrogen bonding.The mRNA coding for the receptor has a nucleotide sequence identical tothe sense strand, with the expected substitution of thymidine byuridine. Thus, based upon the knowledge of the receptor sequence,synthetic oligonucleotides can be synthesized. These oligonucleotidescan bind to the DNA and RNA coding for the receptor. The activefragments of the invention, which are complementary to mRNA and thecoding strand of DNA, are usually at least about 15 nucleotides, moreusually at least 20 nucleotides, preferably 30 nucleotides and morepreferably may be 50 nucleotides or more. The binding strength betweenthe sense and antisense strands is dependent upon the total hydrogenbonds. Therefore, based upon the total number of bases in the mRNA, theoptimal length of the oligonucleotide sequence may be easily calculatedby the skilled artisan.

[0106] The sequence may be complementary to any portion of the sequenceof the mRNA, i.e., it may be proximal to the 5′-terminus or cappingsite, or downstream from the capping site, between the capping site andthe initiation codon and may cover all or only a portion of thenon-coding region or the coding region. The particular site(s) to whichthe antisense sequence binds will vary depending upon the degree ofinhibition desired, the uniqueness of the sequence, the stability of theantisense sequence, etc.

[0107] In the practice of the invention, expression of the IL-9 receptoris down-regulated by administering an effective amount of syntheticantisense oligonucleotide sequences described above. The oligonucleotidecompounds of the invention bind to the mRNA coding for human IL-9 orIL-9 receptors thereby inhibiting expression (translation) of theseproteins. See Gruss et al., “Interleukin 9 is expressed by primary andcultural Hodgkin and Reed-Sternberg cells.” Cancer Res. 52:1026-31 (Feb.15, 1992) The isolated DNA sequences containing various mutations suchas point mutations, insertions, deletions, or spliced mutations of IL-9are useful in gene therapy as well.

[0108] In addition to the direct regulation of the IL-9 receptor, thisinvention also encompasses methods of downstream regulation whichinvolve inhibition of signal transduction. In particular, a furtherembodiment of this invention is drawn to inhibition of tyrosinephosphorylation. It is known in the art that highly exergonicphosphoryl-transfer reactions are catalyzed by various enzymes known askinases. In other words, a kinase transfers phosphoryl groups betweenATP and a metabolite. IL-9 induces tyrosine phosphorylation of multipleproteins; it is known in the art that in addition to the activation ofJAK1 and JAK3 tyrosine kinases, IL-9 also induces tyrosinephosphorylation of Stat3.⁵⁸ Phoshorylation of Stat3 is unique to theIL-9 signal transduction pathway and hence is a perfect target forinhibitors.⁵⁸ This invention includes within its scope tyrphostins whichare specific inhibitors of protein tyrosine kinases. Thus, tyrphostins(obtained for example from Calbiochem) and other similar inhibitors ofthese kinases are useful in the modulation of signal transduction andare useful in the treatment of atopic allergies and asthma.

[0109] In still another aspect of the invention, it was surprisingly,found that aminosterol compounds are also useful in the inhibition ofsignal transduction due to IL-9 stimulation. Aminosterol compounds whichare useful in this invention are described in U.S. patent application,Ser. No. 08/290,826 and its related applications Ser. Nos. 08/416,883and 08/478,763 as well as in 08/483,059 and its related application Ser.Nos 08/483,057, 08/479,455, 08/479,457, 08/475,572, 08/476,855,08/474,799 and 08/487,443, which are specifically incorporated herein byreference.

[0110] In addition, the invention includes pharmaceutical compositionscomprising the compounds of the invention together with apharmaceutically acceptable carrier. Pharmaceutically acceptablecarriers can be sterile liquids, such as water and oils, including thoseof petroleum, animal, vegetable or synthetic origin, such as peanut oil,soybean oil, mineral oil, sesame oil and the like. Water is a preferredcarrier when the pharmaceutical composition is administeredintravenously. Saline solutions and aqueous dextrose and glycerolsolutions can also be employed as liquid carriers, particularly forinjectionable solutions. Suitable pharmaceutical carriers are describedin Martin, E. W., Remington's Pharmaceutical Sciences, specificallyincorporated herein by reference.

[0111] The compounds used in the method of treatment of this inventionmay be administered systemically or topically, depending on suchconsiderations as the condition to be treated, need for site-specifictreatment, quantity of drug to be administered, and similarconsiderations.

[0112] Topical administration may be used. Any common topical formationsuch as a solution, suspension, gel, ointment, or salve and the like maybe employed. Preparation of such topical formulations as are welldescribed in the art of pharmaceutical formulations as exemplified, forexample, by Remington's Pharmaceutical Science, Edition 17, MackPublishing Company, Easton, Pa. For topical application, these compoundscould also be administered as a powder or spray, particularly in aerosolform. The active ingredient may be administered in pharmaceuticalcompositions adapted for systemic administration. As is known, if a drugis to be a administered systemically, it may be confected as a powder,pill, tablets or the like, or as a syrup or elixir for oraladministration. For intravenous, intraperitoneal or intra-lesionaladministration, the compound will be prepared as a solution orsuspension capable of being administered by injection. In certain cases,it may be useful to formulate these compounds in suppository form or asan extended release formulation for deposit under the skin orintermuscular injection. In a preferred embodiment, the compounds ofthis invention be administered by inhalation. For inhalation therapy thecompound may be in a solution useful for administration by metered doseinhalers, or in a form suitable for a dry powder inhaler.

[0113] An effective amount is that amount which will down regulateeither the expression of IL-9 or the functions controlled by IL-9. Agiven effective amount will vary from condition to condition and incertain instances may vary with the severity of the condition beingtreated and the patient's susceptibility to treatment. Accordingly, agiven effective amount will be best determined at the time and placethrough routine experimentation. However, it is anticipated that in thetreatment of asthma-related disorders in accordance with the presentinvention, a formulation containing between 0.001 and 5 percent byweight, preferably about 0.01 to 1%, will usually constitute atherapeutically effective amount. When administered systemically, anamount between 0.01 and 100 mg per kg body weight per day, butpreferably about 0.1 to 10 mg/kg, will effect a therapeutic result inmost instances.

[0114] Applicant also provides for a method to screen for the compoundsthat down regulate the expression of IL-9 or the functions controlled byIL-9. One may determine whether the functions expressed by IL-9 aredown-regulated using techniques standard in the art.⁵⁷⁻⁶⁰ In a specificembodiment, applicant provides for a method of identifying compoundswith functions comparable to Met IL-9. Thus, in one embodiment, serumtotal IgE may be measured using techniques well known in the art⁴² toassess the efficacy of a compound in down regulating the functions ofIL-9 in vivo. In another embodiment, bronchial hyperresponsiveness,bronchoalveolar lavage, and eosinophilia may be measured usingtechniques well known in the art⁴² to assess the efficacy of a compoundin down regulating the functions of IL-9 in vivo. In yet anotherembodiment, the functions of IL-9 may be assessed in vitro. As is knownto those in the art, human IL-9 specifically induces the rapid andtransient tyrosine phosphorylation of multiple proteins in MO7e cells.The tyrosine phosphorylation of Stat3 transcriptional factor appears tobe specifically related to the actions of IL-9. Another method tocharacterize the function of IL-9 and IL-9-like molecules that dependson the “stable expression” of the IL-9 receptor uses the well knownmurine TS1 clones to assess human IL-9 function with a cellularproliferation assay.

[0115] The invention also includes a simple screening assay forsaturable and specific ligand binding based on cell lines that expressthe IL-9 receptor.^(46,59) The IL-9 receptor is expressed in on a widevariety of cell types, including K562, C8166-45,B cells, T cells, mastcells, neutrophils, megakaryocytes (UT-7 cells),⁵³ the humanmegakaryoblastic leukemia cell lines MO7e⁵⁷, TF1,⁵⁹ macrophages, fetalthymocytes, the human kidney cell line 293,⁵³ and murine embryonichippocampal progenitor cell lines.^(46,52,53) In another embodiment,soluble IL-9 receptor may be used to evaluate ligand binding andpotential receptor antagonists.

[0116] The practice of the present invention will employ theconventional terms and techniques of molecular biology, pharmacology,immunology, and biochemistry that are within the ordinary skill of thosein the art. See, for example, Sambrook et al., MOLECULAR CLONING: ALABORATORY MANUAL, 2d ed. Cold Spring Harbor Laboratory Press [19851, orAusubel et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley &Sons, Inc. [1994].

[0117] Nonetheless, we offer the following basic background information.The body's genetic material, or DNA, is arranged on 46 chromosomes,which each comprises two arms joined by a centromere. Each chromosome isdivided into segments designated p or q. The symbol p is used toidentify the short arm of a chromosome, as measured from the centromereto the nearest telomere. The long arm of a chromosome is designated bythe symbol q. Location on a chromosome is provided by the chromosome'snumber (i.e., chromosome 5) as well as the coordinates of the p or qregion (i.e., q31-q33). In addition, the body bears the sex chromosomes,X and Y. During meiosis, the X and Y chromosomes exchange DNA sequenceinformation in areas known as the pseudoautosomal regions.

[0118] DNA, deoxyribonucleic acid, consists of two complementary strandsof nucleotides, which include the four different base compounds, adenine[A], thymine [T], cytosine [C], and guanine [G]. A of one strand bondswith T of the other strand while C of one strand bonds to G of the otherto form complementary “base pairs,” each pair having one base in eachstrand.

[0119] A sequential grouping of three nucleotides [a “codon”] codes forone amino acid. Thus, for example, the three nucleotides CAG codes forthe amino acid Glutamine. The 20 naturally occurring amino acids, andtheir one letter codes, are as follows: Alanine Ala A Arginine Arg RAsparagine Asn N Aspartic Acid Asp D Asparagine or Aspartic acid Asx BCysteine Cys C Glutamine Gln Q Glutamine Acid Glu E Glutamine orGlutamic acid Glx Z Glycine Gly G Histidine His H Isoleucine Ile ILeucine Leu L Lysine Lys K Methionine Met M Phenylalanine Phe F ProlinePro P Serine Ser S Threonine Thr T Tryptophan Trp W Tyrosine Tyr YValine Val V

[0120] Amino acids comprise proteins. Amino acids may be hydrophilic,i.e., displaying an affinity for water, or hydrophobic, i.e., having anaversion to water. Thus, the amino acids designated as G, A, V, L, I, P,F, Y, W, C and M are hydrophobic and the amino acids designated as S, Q,K, R, H, D, E, N and T are hydrophilic. In general, the hydrophilic orhydrophobic nature of amino acids affects the folding of a peptidechain, and consequently the three dimensional structure of a protein.

[0121] DNA is related to protein as follows:

[0122] Genomic DNA comprises all the DNA sequences found in anorganism's cell. It is “transcribed” into messenger RNA [“mRNA”].Complementary DNA [“cDNA”] is a complementary copy of mRNA made byreverse transcription of mRNA. Unlike genomic DNA, both mRNA and cDNAcontain only the protein-encoding or polypeptide-encoding regions of theDNA, the so-called “exons.” Genomic DNA may also include “introns,”which do not encode proteins.

[0123] In fact, eukaryotic genes are discontinuous with proteins encodedby them, consisting of exons interrupted by introns. After transcriptioninto RNA, the introns are removed by splicing to generate the maturemessenger RNA (mRNA). The splice points between exons are typicallydetermined by consensus sequences that act as signals for the splicingprocess. Splicing consists of a deletion of the intron from the primaryRNA transcript and a joining or fusion of the ends of the remaining RNAon either side of the excised intron. Presence or absence of introns,the composition of introns, and number of introns per gene, may varyamong strains of the same species, and among species having the samebasic functional gene. Although in most cases, introns are assumed to benonessential and benign, their categorization is not absolute. Forexample, an intron of one gene can represent an exon of another. In somecases, alternate or different patterns of splicing can generatedifferent proteins from the same single stretch of DNA. In fact,structural features of introns and the underlying splicing mechanismsform the basis for classification of different kinds of introns.

[0124] As to the exons, these can correspond to discrete domains ormotifs, as for example, functional domains, folding regions, orstructural elements of a protein; or to short polypeptide sequences,such as reverse turns, loops, glycosylation signals and other signalsequences, or unstructured polypeptide linker regions. The exon modulesof the present combinatorial method can comprise nucleic acid sequencescorresponding to naturally occurring exon sequences or naturallyoccurring exon sequences which have been mutated (e.g. point mutations,truncations, fusions).

[0125] Returning now to the manipulation of DNA, DNA can be cut,spliced, and otherwise manipulated using “restriction enzymes” that cutDNA at certain known sites and DNA ligases that join DNA. Suchtechniques are well known to those of ordinary skill in the art, as setforth in texts such as Sambrook, et al., MOLECULAR CLONING: A LABORATORYMANUAL, 2d ed. Cold Spring Harbor Laboratory Press [1985] or Ausubel etal., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, Inc.[1994].

[0126] DNA of a specific size and sequence can then be inserted into a“replicon,” which is any genetic element, such as a plasmid, cosmid, orvirus, that is capable of replication under its own control. A“recombinant vector” or “expression vector” is a replicon into which aDNA segment is inserted so as to allow for expression of the DNA, i.e.,production of the protein encoded by the DNA. Expression vectors may beconstructed in the laboratory, obtained from other laboratories, orpurchased from commercial sources.

[0127] The recombinant vector [known by various terms in the art] may beintroduced into a host by a process generically known as“transformation.” Transformation means the transfer of an exogenous DNAsegment by any of a number of methods, including infection, directuptake, transduction, F-mating, microinjection, or electroporation intoa host cell.

[0128] Unicellular host cells, known variously as recombinant hostcells, cells, and cell culture, include bacteria, yeast, insect cells,plant cells, mammalian cells and human cells. In particularly preferredembodiments, the host cells include E.coli, Pseudonas, Bacillis,Streptomyces, Yeast, CHO, R1-1, B-W, LH, COS-J, COS-7, BSC1, BSC40,BMT10, and S69 cells. Yeast cells especially include Saccharomyces,Pichia, Candida, Hansenula, and Torulopis.

[0129] As those skilled in the art recognize, the expression of the DNAsegment by the host cell requires the appropriate regulatory sequencesor elements. The regulatory sequences vary according to the host cellemployed, but include, for example, in prokaryotes, a promoter,ribosomal binding site, and/or a transcription termination site. Ineukaryotes, such regulatory sequences include a promoter and/or atranscription termination site. As those in the art well recognized,expression of the polypeptide may be enhanced, i.e., increased over thestandard levels, by careful selection and placement of these regulatorysequences.

[0130] In other embodiments, promoters that may be used include thehuman cytomegalovierus (CMV) promoter, tetracycline inducible promoter,simian virus (SV40) promoter, moloney murine leukemia long terminalrepeat (LTR) promoter, glucocorticoid inducible murine mammary tumorvirus (MMTV) promoter, Herpes thymidine kinase promoter, murine andhuman β-actin promoters, HTLV1 and HIV IL-9 5′ flanking region, humanand mouse IL-9 receptor 5′ flanking region, bacterial tac promoter anddrosophila heat shock scaffold attachment region (SAR) enhancerelements.

[0131] The DNA may be expressed as a polypeptide of any length such aspeptides, oligopeptides, and proteins. Polypeptides also includetranslational modifications such as glycosylations, acetylations,phosphorylations, and the like.

[0132] Another molecular biologic technique of interest to the presentinvention is “linkage analysis.” Linkage analysis is an analytic methodused to identify the chromosome or chromosomal region that correlateswith a trait or disorder.⁴⁴ Chromosomes are the basic units ofinheritance on which genes are organized. In addition to genes, artisanshave identified “DNA markers” on chromosomes. DNA markers are knownsequences of DNA whose identity and sequence can be readily determined.Linkage analysis methodology has been applied to the mapping of diseasegenes, for example, genes relating to susceptibility to asthma, tospecific chromosomes.^(42,44)

[0133] Other embodiments of the invention will be apparent to thoseskilled in the art from consideration of the specification and practiceof the invention disclosed. It is intended that the specifications andexamples be considered exemplary only with a true scope of the inventionbeing indicated by the claims.

Methods

[0134] In conducting the experiments described in the Examples below,applicant used the following methods:

Patient Populations

[0135] Asthma families were recruited from two sources.^(27,42,79-83,88)In each case patients were genotyped with respect to the polymorphism atposition 3365 of the human IL-9 gene [GenBank accession number M30136].

[0136] A third population of 74 individuals was ascertained randomlywith respect to asthma and atopy from the East Coast of the UnitedStates. The frequency of the Met substitution at codon 117 was used asan unbiased estimate of the prevalence of this variant in the generalpopulation.

[0137] A fourth population of 49 individuals was ascertained randomlywith respect to asthma and atopy from the Philadelphia, Pennsylvaniaarea. Total serum IgE were assayed by enzyme-linked immunosorbent test[ELISA, Genzyme, Cambridge, Mass.]. DNA was extracted from the WBC inperipheral blood from each individual. Analyses of genetic markers(genotyping) and candidate genes were performed on the genomic DNAextracted. Once again, the frequency of the Met substitution at codon117 was used as an unbiased estimate of the prevalence of this variantin the general population.

Oligonucleotide Primers

[0138] All primers were designed using OLIGO 4.0. Characterization ofthe IL-9 gene was carried out using primers surrounding each of the 5exons of the reported sequence. The primer sequences surrounding eachexon were: exon 1 [upper] [5′ GCT CCA GTC CGC TGT CAA 3′] and [lower][5′ CTC CCC CTG CAG CCT ACC 3′][product size 150 bp]; exon 2 [upper] [5′CGG GGC TGA CTA AAG GTT CT 3′] and [lower] [5′ GTT CTT AAA GAG CAT TCACT 3′][product size 99 bp]; exon 3 [upper] [5′ ATT TTC ACA TCT GGA ATCTTC ACT 3′] and [lower][5′ AAT. CCA AGG TCA ACA TTA TG 3′][product size113 bp]; exon 4 [upper] [5′ TTT CTT TGA ATA AAT CCT TAC 3′] and [lower][5′ GAA ATC ACC AAC AGG AAC ATA 3′][product size 206 bp]; and exon 5[upper] [5′ATC AAC TTT CAT CCC CAC AGT 3′] and [lower] [5′ GGA TAA ATAATA TTT CAT CTT CAT 3′]. Each exon was examined first by a single strandconformational polymorphism assay [SSCP].^(72,77) The primers for exon 5produced a 160 bp product after polymerase chain reaction [PCR]amplification which was also examined by direct solid phase sequenceanalysis.^(72,77) The upper primer was synthesized with a 5′ biotinlabel and, following amplification, the PCR product was captured by astreptavidin-linked paramagnetic bead [Dynal] and characterized bySanger sequencing as described elsewhere.⁷⁷ Sequence polymorphisms weredistinguished from artifact by repeated analyses.

SSCP Analysis

[0139] SSCP, a method for detection of polymorphisms on the basis ofchanges in migration of single-stranded DNA exposed to an electricfield,⁷² was carried out as set forth in Schwengel et al., (1994) atroom temperature with and without 10% glycerol using 6% polyacrylamidegel electrophoresis at a cross-linking monomer concentration of 2.67%.⁷⁷Four μl of PCR product was mixed with 5 μl 2× stop buffer [95%formamide, 20 mM EDTA, 0.05% BPB, 0.05% xylene cyanol], and 1 μl 0.5%SDS and 50 μM EDTA, denatured at 85-90 oC for 8 minutes, and thenimmediately placed on ice. Electrophoresis was carried out at 12 wattsfor approximately 24 hours for glycerol containing gels and 12 hours fornon-glycerol gels. The gels were then dried and exposed to Kodak XAR®film.

DNA Sequencing

[0140] Direct DNA sequencing of the PCR products was accomplished usingsolid phase techniques after verifying the presence of the correct sizePCR product on a 1% agarose gel stained with ethidium bromide as setforth in Schwengel et al., 1994.⁷⁷ Twenty μl of PCR product wasincubated with 40 μl of Dynabeads® m-280 [Dynal] for 15 minutes. Thebeads were washed and diluted as suggested by the manufacturer. Eachsample was subsequently washed with B&W buffer containing 10 mM tris-HClpH 7.5, 1 mM EDTA, 2 M NaCl, denatured with 0.1 N NaOH, and then washedwith 0.1 N NaOH, B&W buffer, and 10 mM Tris-HCl pH 8 and 1 mM EDTA [TE].The pellet of beads was resuspended with 10 μl of H2O.

[0141] Sanger sequencing reactions were carried out using Sequenase[United States Biochemical Co.]. ³⁵S-DATP or ³³P-DATP was incorporatedinto the sequencing reactions, and the products were electrophoresedthrough either 5? or 6% polyacrylamide gels containing 7 M urea. Gelswere dried without fixing and exposed to X-ray film. Alleles weredetermined by comparing the genotypes of parents and offspring.Infrequent artifacts were easily distinguished from true sequencepolymorphisms by repetition.

[0142] DNA was available and extracted from peripheral leukocytes.Genomic DNA was diluted to a concentration of 200 μg/ml foramplification.^(27,42) Simple sequence repeats [SSR] including DXYS154were selected from the Genome Data Base [GDB; Welch library, JohnsHopkins University, Baltimore, Md.]. Genotyping of the sKK-1 marker wascarried out using the following primers sKK-1U [5′ CAA ATC TGA AGA GCAAAC TAT 3′] and sKK-1L [5′ TTA AAA AAT TCA TTT CAG TAT TCT 3′] whichproduce a 90 bp product. Each SSR product was amplified by PCR⁷² andsized according to methods previously described.^(27,42) Sample handlingwas carried out as described by Weber et al. with minormodifications.^(71,27,42) Genotypes were determined from two independentreadings of each autoradiograph. Individuals genotyping the familieswere blinded to the clinical data.

RFLP Analysis

[0143] As a result of the C to T polymorphism at position 3365, a StyIrestriction fragment length polymorphism [RFLP] was produced at position52 of the IL-9 exon 5 PCR product. To test for the presence of this DNAsequence variant the lower primer from exon 5 was end-labeled prior toPCR amplification. The PCR product was then digested with StyI producingtwo fragments 108 bp [labeled] and 52 bp [unlabeled] in length. ThisRFLP was used along with SSCP to confirm the presence of thispolymorphism in families and individuals.

Linkage Analyses and Data Management

[0144] Linkage analyses were performed using affected sib-pair methods[SIBPAL, S.A.G.E.],⁷⁸ an established approach for the investigation ofthe genetic basis of complex traits, such as BHR, atopy, and asthma.Affected sib-pairs are usually tested first, since a proportion ofunaffected sib-pairs may still be gene carriers but do not express thetrait. In contrast to LOD score methods where the model of inheritance[dominant, recessive, etc.] must be specified exactly, analysis bysib-pair methods makes no explicit assumptions in this regard. Thus, insib-pair analyses the parents' clinical information is not used intesting for linkage. The pertinent observation in these methods is howoften two affected offspring share copies of the same parental markerallele.⁴⁴ If the same copy of a parental marker allele is observed indifferent offspring, they are said to be inherited “identical bydescent.” Linkage is suggested when affected sib-pairs are identical bydescent for a marker allele significantly more often than expected bychance [50%]. When the same marker allele is transmitted with thedisease gene in different offspring, this implies that the marker locusis linked, or must be located close enough on the same chromosome, tothe disease gene so they cosegregate during meiosis. The trait is thenmapped by knowing the chromosomal localization of the marker.

[0145] Linkage in humans may also be established by the method oflikelihood ratios. This method involves comparison of the probabilitythat observed family data would arise under one hypothesis, forinstance, linkage between two DNA markers, to the probability that itwould arise under an alternative hypothesis, typically, nonlinkage. Theratio of these probabilities is called the odds ratio for one hypothesisrelative to the other. By convention, mammalian geneticists prefer thelog of the odds ration, or the LOD score. Generally, linkage isconsidered proved when the odds in favor of linkage versus nonlinkagebecome overwhelming, or reach 1,000:1 [LOD=3]. Linkage is rejected whenthe odds drop to 100:1 against this hypothesis [LOD=−2]. The maximumlikelihood estimate is the recombination fraction where the likelihoodratio is largest. LODs from multiple pedigrees are thus added until thescore grows to 3 [signifying 1,000:1 odds] or falls to −2 (indicating100:1 odds].

[0146] All clinical and genotype data is managed using EXCELL® on aMacIntosh® or Sun Microsystems® computer. Statistical analyses werepreformed using JMP [SAS Institute, Inc. Cary, N.C.]. TheWilcoxon/Kruskal-Wallis Tests [rank sums] was used to test whetherindividuals who were homozygous [Met/Met], heterozygous [Met/Thr], orhomozygous [Thr/Thr] at codon 117 differ in their serum total IgE. AllP-values are two-tailed except affected sib-pair analyses, where aone-tailed test was used because only an increased sharing of alleleswas expected.

[0147] Having provided this background information, applicant nowdescribes preferred aspects of the invention.

EXAMPLE 1 Linkage Analysis Between BHR and Murine Chromosome 13

[0148] As an aid in dissecting the complex genetic determinants of BHR,applicant has developed murine models that differ in their geneticsusceptibility to various bronchoconstrictor stimuli. Inbred animalmodels using recombinant inbred strains [BXD] can facilitate ongoingstudies in humans to determine the number of genes regulatingsusceptibility to BHR, the magnitude of their affect, and their precisechromosomal location. In particular, localizing in an animal model agene determining susceptibility to a critical risk factor for asthma mayaid in the positional cloning of this gene in humans.

[0149] Although the gene[s] predisposing to BHR and atopy had not yetbeen identified prior to this invention, chromosome 5q31-q33 was knownto be syntenic with portions of mouse chromosomes 11, 13, and 18. FIG. 2illustrates the syntenic regions containing numerous positionalcandidates that may potentially play a role in airway inflammationassociated with BHR, atopy, and asthma. Specifically, the region ofhuman chromosome 5q31-q33 demonstrating significant evidence for linkagewith BHR is homologous to portions of mouse chromosomes 11, 13, and 18which contain numerous candidate genes.⁸⁴

[0150] In particular, IL-9 or a nearby gene have recently been suggestedas likely candidates on the basis of linkage disequilibrium between logserum total IgE levels and a marker in this gene using a randomlyascertained population.⁴³

[0151] Despite comparisons with four candidate intervals, evidence forlinkage was found for only one region, designated Aib1 [atracuriuminduced bronchoconstriction 1]. FIG. 3 provides the results.Specifically, FIG. 3 sets forth the LOD score curve on mouse chromosome13 for atracurium-induced airway responsiveness in 24 BXD RI strainswhich are derived from the hyporesponsive C57BL/6J and thehyperresponsive DBA/2J progenitor strains [solid line]. The LOD scorecurve resulting from the selective genotyping of 20 BXD strains is alsoshown [dashed line]. BXD strains −2, -6, -18, and -32 were not used inthe second analysis since they were intermediate in phenotype displayinga mean response greater than 1 standard deviation below the DBA/2 andabove the C57BL/6 mean responses. The bronchoconstrictor response toatracurium, 20 mg/kg given intravenously, was assessed by the change inpeak inspiratory pressure integrated over time [4 min], termed theairway pressure time index [APTI]. Atracurium-induced APTI was measuredin 2-8 animals per RI strain. Marker data were obtained from the RWEdata base in the Map Manager data analysis program. The genetic distance[cM] between markers is indicated on the abscissa. LOD scores werecalculated by the MAPMAKER/QTL linkage program. A QTL was detected inthis region and termed atracurium-induced bronchoconstriction-1 [Aib1].

[0152]FIG. 3 indicates that this quantitative trait locus [QTL] islocated on the midportion of murine chromosome 13 and attained thisinterval a maximum likelihood log of the odds [LOD] of 2.42. Forty-fourpercent of the total variance in atracurium-induced bronchoconstrictionwas explained at Aib1 when all of the markers in the BXD map wereanalyzed. The LOD for chromosome 13 increased to 2.85 when QTL analyseswere run after excluding the four strains [BXD-2, -6, -18, and -32] thatwere intermediate responders to atracurium. The known positionalcandidates in the linked region of chromosome 13 include: D1 dopaminereceptor [Drd1], fibroblast growth factor receptor 4 [Fgfr4], lymphocyteantigen-28 [Ly28], thiopurine methyltransferase [Tpmt], and IL-9.

[0153] Because the applicant was specifically testing for linkage tofour candidate regions in the mouse, based on previous mapping data inthe human, the data presented here are highly significant. As stated inthe classic paper by Lander and Botstein,⁶⁷ a false positive rate forlinkage will result if the LOD threshold (T)is chosen so that T=½(log10e)(Z α/n)2 (where n is equal to the number of intervals tested).Typically a minimum LOD of 3.3 is required as evidence of linkage.⁶⁷However, this threshold is based on the assumption that one is searchingthe entire genome. These same authors point out that a LOD of 0.83 issufficient when only one region is examined. In this case, with fourcandidate regions, a P value (α) of 0.0125 for each region is requiredto obtain a true P≦0.05, when one corrects for multiple independentcomparisons. Adopting a 5% error rate that even a single false positivefinding will occur, as suggested by Soller and Brody,⁶⁸ and solving theequation ½(log10 e)(Z α/n) 2 , yields a LOD threshold of at least 1.36.A maximal LOD of 1.48 was obtained for the Il-9 gene candidate.Restricting the acceptable false positive error rate to ≦0.1%, increasesthe LOD threshold to 2.36. Thus, the maximal LOD generated of 2.42 forthe candidate interval on chromosome 13 (Aib1) is highly significant.

[0154] These LOD threshold data provide evidence of a conserved linkagefor BHR in humans and mice. BHR in humans links to the region onchromosome 5q containing a number of growth factors and cytokinesincluding the IL-9 gene and the Aib1 locus maps to the IL-9 region ofmurine chromosome 13.

EXAMPLE 2 Identification of an IL-9 Gene Polymorphism

[0155] Applicant demonstrated conserved linkage between the mouse andhumans for BHR. These data suggest that variation in the functions ofthis gene or DNA sequence may be important in regulating bronchialresponsiveness in the mouse. Using the methods described above, a uniqueproduct of the correct size was identified by gel electrophoresis foreach of the exons of human IL-9 after PCR. A single polymorphism wasidentified by SSCP in exon 5 of the human IL-9 gene. Direct DNA sequenceanalysis demonstrated a C to T nucleotide substitution at position 3365[GenBank accession number M30136] of the human IL-9 gene as the cause ofthe novel SSCP conformer. This DNA sequence change predicts anonconservative substitution of a methionine [hydrophobic] for athreonine [hydrophilic] at amino acid 117 of the IL-9 protein.

[0156] Exon 5 codes for this segment of the protein which is within themost highly conserved interval of human IL-9 as compared to the mouseIL-9 sequence (see FIG. 4).

[0157] Individuals were genotyped from various populations to examinethe frequency of these alleles by direct analyses of the nucleotidesubstitution in the coding sequence of human IL-9. Two of 394individuals from a group of asthmatic families were homozygous [Met/Met]at codon 117 [0.5%]. There were 91 [23.1%] heterozygous, and 301 [76.4%]homozygous [Thr/Thr] individuals. The true prevalence for this IL-9variant is likely to be significantly higher because the Italianpopulation of families was ascertained through symptomatic patients withasthma. From a separate ethnically diverse population ascertainedrandomly with respect to atopy and asthma, there were 1 of 49individuals homozygous for [Met/Met] at codon 117 [, 2.0%]. There were11 [22.4%] heterozygous, and 37 [75.5%] homozygous [Thr/Thr]individuals. The prevalence of the Met/Thr heterozygotes was 18.9% in afourth population ascertained randomly with respect to atopy and asthma.Thus, approximately 20% of the population are likely to representcarriers of the T allele at position 3,365 as compared to the reportedsequence [GenBank accession number M30136]. Because it is well known inthe art that the frequency of any allele in the population is p2+2pq+q2,then, approximately 4% of the population is expected to be Met/Methomozygous at codon 117 of IL-9.

[0158] Overall, serum total IgE averaged 44.5 I.U. for homozygousindividuals [Met/Met], which was significantly different from those whowere homozygous wild type [Thr/Thr] [351.7I.U.], or heterozygous[Met/Thr] [320.9 I.U.]. See FIG. 5. The homozygous protected individuals[Met/Met] failed to demonstrate evidence of atopic allergy except for asingle positive skin test in one individual. These data indicate thatthis novel DNA polymorphism, when inherited in the homozygous state, isassociated with protection from atopic allergy, including lower serumtotal IgE.

[0159]FIG. 6 illustrates the PCR amplification of the IL-9 simplesequence repeat polymorphism. This marker is compared with genotype forthese individuals for the restriction fragment length polymorphismproduced by the nucleotide polymorphism at position 3,365 as compared tothe reported sequence [GenBank accession number M30136]. Two familiesare shown. The individuals in lanes 1 and 2 are the parents (Thr/Met) ofindividuals in lanes 3 (Met/Thr) and 4 (Met/Met); lanes 5 (Thr/Thr) and6 (Met/Met) are parents for offspring in lanes 7 (Met/Thr) and 8(Met/Thr). The smallest allele for the IL-9 simple sequence repeatpolymorphism (the lowest band in each figure is 248 nucleotides inlength) is in complete linkage disequilibrium with the Met117 allele(nucleotide substitution at position 3,365 as compared to the reportedsequence [GenBank accession number M30136]) in these individuals and inall individuals from populations tested world wide. This was true inboth the Italian and all random ascertained ethnically diverseindividuals studied, and therefore, this marker may be useddiagnostically to detect the presence of the Met117 allele. These dataare most consistent with the hypothesis that this variant is widelydistributed in populations worldwide and arose before many of thesepopulations separated.

EXAMPLE 3 IL-9 Receptor Expression and Ligand Binding Assay

[0160] Purified recombinant Thr IL-9, Met IL-9, and compoundspotentially resembling Met IL-9 in structure or function areradiolabelled using the Bolton and Hunter reagent as described in BoltonA E, and Hunter W M, Biochem J. 133:529-539(1973). This material islabeled to high specific activity of 2,300 cpm/fmol or greater. HumanK562 and MO7e cells are grown and resuspended at 30° C. in 0.8 ml ofDulbecco's modified Eagle's medium supplemented with 10% (vol/vol) fetalbovine serum, 50 mM 2-mercaptoethanol, 0.55 mM L-arginine, 0.24 mML-asparagine, and 1.25 mM L-glutamine. K562 or MO7e cells are used as isor after transfection with the IL-9 receptor gene as described below.Plasmid DNA containing the full length IL-9 receptor is cloned intopRC/RSV plasmid (In Vitrogen, San Diego) and purified by centrifugationthrough CsCl2. Plasmid DNA (50 micrograms) is added to the cells in 0.4cm cuvettes just before electroporation. After a double electric pulse(750V/74 ohms/40 microFaradays and 100 V/74 ohms/2100 microFaradays) thecells are immediately diluted in fresh medium supplemented with IL-9.After 24 h the cells are washed and incubated in G418 (2.5 mg/ml, GIBCO)with either no ligand, or various concentrations of 125I-labeled ligandat 20° C. for 3 h. An excess of unlabeled ligand is used in parallelexperiments to estimate nonspecific binding. The cells are then washed,filtered, and collected for counting. Specific incorporation iscalculated by Scatchard analysis. Similar competitive assays are runusing 125I-labeled Thr117 IL-9 and various amounts of putative coldligands to assess specific binding.

[0161] Soluble IL-9 receptor including amino acids 44 to 270 (R&DSystems) was incubated with different forms of human recombinant IL-9.Varying amounts of FlagMet117 and FlagThr117 (described in Example 7)were incubated in PBS at room temperature for 30 minutes with 0.5 Ags ofsoluble receptor. EBC buffer (50 mM Tris pH 7.5; 0.1 M NaCl; 0.5% NP40)was added (300 μl) was added along with 1 μg of anti-FLAG monoclonalantibody (IBI) and incubated for 1 hour on ice. Forty microlitres ofprotein A sepharose solution was added to each sample and mixed for 1hour at 4° C. Samples were centrifuged for 1 minute 11,000×G and pelletswere washed 4 times with 500 μl of EBC. Pellets were dissolved in 26 μlof 2×SDS buffer, boiled for 4 minutes, and electrophoresed through an18% SDS polyacrylamide gel. Western blots were performed as described inExample 15 except the blots were probed with an anti-IL-9 receptorantibody (R&D Systems).

[0162]FIG. 25 demonstrates the binding of the IL-9 recombinant proteinssoluble IL-9 receptor. Lane 1 is molecular weight markers, lane 2 is theIL-9 FlagMet117 incubated with the receptor, lane 3 is the IL-9FlagThr117 incubated with the receptor. These data demonstrate that bothforms of the recombinant IL-9 protein are bound to the soluble receptor.Moreover, these data along with those of Example 2 (where hetrozygotesdo not differ in serum Ig-E from homozygous Thr117 individuals) areconsistent with the IL-9 Met117 form representing a weak agonist.

EXAMPLE 4 IL-9 Receptor Expression and Ligand Functional Assay in K562.C8166-45. and MO7e Cells

[0163] Recombinant Thr117 IL-9, Met117 IL-9, and compounds potentiallyresembling Met IL-9 in structure or function were purified and preparedfor use in Dulbecco's modified Eagle's medium. K562, C8166-45 or MO7ecells are used as is or after transfection with the IL-9 receptor geneas described in Example 3. After 24 h of deprivation from growth factorsthe cells are incubated without (control) or with variable amounts ofpurified Thr117 IL-9, Met117 IL-9, and compounds potentially resemblingMet117 IL-9 in structure or function. Cellular proliferation is assessedby measuring acid phosphotase activity. Briefly, quadruplicate samplesof MO7e cells are cultured in flat-bottom microtiter plates (150 or 200microliter wells) with or without ligand for 72 to 96 hours at 37degrees C. Acid phosphatase is measured as suggested by the manufacturer(Clontech, Palo Alto, Calif.). All experiments are repeated at leasttwice.

EXAMPLE 5 Cell Isolation and Culture

[0164] Human peripheral blood mononuclear cells {PBMC} were isolatedfrom healthy donors by density gradient centrifugation using endotoxintested Ficoll-Paque PLUS according to the manufacturer (PharmaciaBiotech, AB Uppsala Sweden). PBMC (5×10⁶), mouse spleen cells (5'10⁶),or 5×10⁶ MO7e cells were cultured in 7 ml of RPMI-1640 (BethesdaResearch Labs (BRL), Bethesda, Md.) supplemented to a finalconcentration of 10% with either isogenic human serum orheat-inactivated FBS. Cells were cultured for 24 hrs at 37° C. eitherunstimulated, or stimulated with either PMA 5 μg/ml/PHA 5 μg/ml, or PHA5 μg/ml/rhIL2 50U (R&D Systems, Minneapolis, Minn.).

EXAMPLE 6 RNA Isolations, RT-PCR, Cloning and Sequencing of RT-PCRProducts

[0165] Total cellular RNA was extracted after 24 hrs from cultured PBMC,mouse spleen cells, and MO7e cells using RNA PCR corekit (Perkin-ElmerCorp, Foster City, Calif.) according to the supplier. One μg of RNA fromeach source was denatured for 5 minutes at 65° C. and then reversetranscribed into cDNA using a 20 μl reaction mixture (RNA PCR corekit,Perkin-elmer Corp, Foster City, Calif.) containing 50 U of MuMLV ReverseTranscriptase, 1 U/μl RNAse Inhibitor, 2.5 mM oligo d(T)16 primer, 1 mMeach of DATP, dCTP, dGTP, dTTP, 50 mM KCl, 10 mM Tris-HCL, pH 7.0, 25 mMMgCl2. The reaction mixture was pipetted into thermocycler tubes, placedin a PCR thermal cycler and subjected to 1 cycle (15 minutes at 42° C.,5 minutes at 99° C., and 5 minutes at 4° C.). A mock reversetranscription reaction was used as a negative control.

[0166] Then this mixture was added to a second tube containing 2 mMMgCl2, 50 mM KCl, 10 mM Tris-HCl, pH 7.0, 65.5 μl of DI water, 2.5 UAmplitaq DNA polymerase, and 1 μl (20 μM) each of oligonucleotidesrepresenting human cDNA IL-9 exon 1 (forward) and exon 5 (reverse), fora final volume of 100 μl. The reaction mixture was subjected to thefollowing PCR conditions: 120 seconds at 98° C., then 30 cycles at: 30seconds at 94° C.; 40 seconds at 55° C; 40 seconds at 72° C. Finally,the reaction mixture was cycled one time for 15 minutes at 72° C. forextension. PCR products representing hIL-9 cDNA were subjected to gelelectrophoresis through 1.5% agarose gels and visualized using ethidiumbromide staining. Products of a mock reverse transcriptase reaction, inwhich H₂O was substituted for RNA, and used as negative controlamplification in all experiments.

[0167] The PCR oligonucleotide primer pairs used in these experiments toamplify cDNA include: human interleukin 9 (hIL-9) exon 1 forward 5′-TCTCGA GCA GGG GTG TCC AAC CTT GGC G-3′ (SEQ ID NO: 1) and exon 5 reverse5′GCA GCT GGG ATA AAT AAT ATT TCA TCT TCA T-3′ (SEQ ID NO: 2); mouseinterleukin 9 (mIL-9) exon 1 forward 5′-TCT CGA GCA GAG ATG CAG CAC CACATG GGG C-3′ (SEQ ID NO: 3) and mouse exon 5 reverse 5′-GCA GCT GGT AACAGT TAT GGA GGG GAG GTT T-3′ (SEQ ID NO: 4); XhoI and PvuII restrictionenzyme recognition sequences are underlined in the human and mouse IL-9primers. PCR products were subcloned into the TA Cloning vector(Invitrogen, San Diego, Calif.). Amplification of the mouse cDNA gave a438 bp product and amplification of the human cDNA gave a 410 bpproduct.

[0168] Complementary DNAs for human IL-9 and murine mIL-9 were generatedand amplified by RT-PCR using IL-9 exon 1 and 5 specific primerscontaining digestion sites for XhoI and PvuII restriction endonucleases.Amplification products for hIL-9 and mIL-9 were isolated from 2.5%agarose gels using silica (Sambrook, J. et al. (1989) Molecular Cloning:A Laboratory Manual Cold Spring Harbor Laboratory Press, New York)(incorporated herein by reference in its entirety). After recovery, thecDNA products were ligated into the TA Cloning vector (Invitrogen Corp.,San Diego, Calif.) and then used to transform INVαF′ competent cells,according to the manufacturer's instructions. Plasmids containing hIL-9and mIL-9 cDNA inserts were isolated by conventional techniques(Sambrook, J. et al. (1989) Molecular Cloning: A Laboratory Manual ColdSpring Harbor Laboratory Press, New York). After amplification the DNAsequence including and surrounding each insert was analyzed forPCR-induced or cloning-induced errors.

[0169] hIL-9 and mIL-9 cDNA inserts were sequenced by thedideoxy-mediated chain termination method (Sanger et al. (1977) Proc.Natl. Acad. Sci. USA 74:5463), using the M13 (-20) forward primer(5′-GTA AAA CGA CGG CCA GT-3′) (SEQ ID NO: 17) and Sequenase™ (USB), andanalyzed by gel electrophoresis (Sambrook, J. et al. (1989) Molecularcloning: a laboratory manual Cold Spring Harbor Laboratory Press, NewYork). hIL-9 and mIL-9 cDNA inserts without cloning and/or Taqpolymerase-induced sequence errors (see translated cDNA sequences FIGS.7 and 8) were subcloned into expression vectors (see FIGS. 9-12) or usedto create missense mutations and deletion mutants.

EXAMPLE 7 Cloning and Expression of IL-9 Constructs In Vitro GeneralCloning Methods for Constructs

[0170] hIL-9 was subcloned into procaryotic expression vectors. TheTA2AAF1 met and thr vectors were digested by EcoRI and the 0.420 kBfragment (containing an XhoI site at the 5′ end of the hIL9 cDNA) wascloned into the EcoRI site contained with the polylinker of pBluescript(PBS) (Stratagene). Clones in the sense orientation to the T3 promoterwere then digested with XhoI (the fragment contained a 5′ XhoI site fromthe IL-9 cDNA insert from TA vectors and a 3′ XhoI site from the PBSpolylinker) and inserts were subcloned into the XhoI sites of theprocaryotic expression vectors PGEX and pFLAG.

Cloning and Expression of IL-9 Constructs in the pGEX-4T-1 Glutathiones-transferase Gene Fusion Vector

[0171] For the expression, purification, and detection of IL-9 protein,IL-9 cDNA inserts were subcloned into the XhoI site within the multiplecloning cassette of the 4.9 Kb pGEX-4T-1 glutathione s-transferase genefusion vector (Pharmacia Biotech, Piscataway, N.J.) by standardtechniques. Briefly, TA clones containing intact IL-9 cDNA sequences,and the pGEX-4T-1 vector were digested for one hour at 37° C. using XhoIand PvuII restriction endonuclease in the presence of 1× React 2 buffer(New England Biolabs, Beverly, Mass.) (total volume 50 μl). Productswere electrophoresed in a 1.5% preparative agarose gel with 10 μg/mlethidium bromide. The appropriate sized DNA band was excised, theagarose was melted at 45° C. for 10 minutes in 3 volumes of NaI stocksolution. A silica matrix solution in DI H₂O (Geneclean II, LaJolla,Calif.) was added to the solution at 5 μl per 5 μg of DNA and 1 μl per0.5 μg of DNA above 5 μg. The slurry was incubated at 4° C. andoccasionally shaken during 30 minutes. The slurry was then pelleted viamicrocentrifugation, washed 3 times in low-salt buffer and resuspendedin 10 μl of DI H₂O to elute the DNA from the silica. A finalmicrocentrifugation provided the 10 μl solution containing purified DNA.

[0172] Products were resuspended in 50 μl of DI H₂O and precipitated bythe addition of 2 volumes of ethanol and 1/10 volume 3M sodium acetate.Samples were centrifuged at RT at 14,000 rpm for 10 minutes, air driedunder negative pressure and resuspended in an appropriate volume of DIH₂O. Ligations and transformations of DH5a bacteria (GIBCO/BRL,Gaithersburg, Md.) with mIL-9 and hIL-9 cDNA inserts in the pGEX-4T-1vector were performed using standard techniques.

[0173] To confirm that the hIL-9 cDNA inserts contained in the pGEX-4T-1vector were of the correct nucleotide sequence, plasmids containingcandidate IL-9 cDNA were sequenced via the dideoxy-mediated chaintermination method using the aforementioned mIL-9 and hIL-9cDNA-specific oligonucleotides (exon 1 forward, exon 5 reverse primers).

[0174] Recombinant fusion proteins were obtained from large scalecultures. The overnight culture of transformed E. coli (50 ml) wasinoculated into fresh LB/amp broth. The culture was incubated for 4 hrat 37° C. with vigorous shaking, isopropyl β-D-thiogalactopyranoside wasthen added to a final concentration of 1 mM, and the culture wasincubated for an additional 1.5 h. The cells were harvested bycentrifugation at 500×g at 4° C. and recombinant variants were purifiedby making use of affinity chromatography on glutathione-sepharose 4Bcolumn (Pharmacia) for GST-fusion proteins. Some variants were expressedas inclusion bodies and were purified from insoluble inclusion bodies bythe procedure described by Marston (1987 The purification of eukaryoticpolypeptides expressed in E.coli in Clover D. M. ed.

[0175] DNA cloning: A practical approach, IRL Press, Oxford, 59-88).Briefly, the cells were lysed with lysozyme followed by treatment withdeoxycholic acid. Contaminating nucleic acids were removed by treatmentwith DNase I. The insoluble material was washed once with 2 M urea andfinally solubilized in lysis buffer (50 mM Tris-Cl, pH 8.0, 1 mM EDTA,100 mM NaCl) containing 8 M urea. The solubilized components from theinclusion bodies were dialyzed stepwise against decreasingconcentrations of urea (starting with 8, 6, 4 and 2 M of urea) in lysisbuffer to allow for refolding of the denatured protein. Finally, thesample was dialyzed against 2 M urea and 2.5% β-mercaptoethanol (β-ME)and centrifuged at 10,000 g for 15 min. The fusion protein was finallydialyzed against 0.01 M Tris-Cl, pH 8.0. Fusion proteins expressed inpGEX-4T vector were cleaved with 100 U of Thrombin for 6 hr at 37° C.and recovered in flow through fractions after chromatography onglutathione-Sepharose 4B column. Final purification was achieved bychromatography on Sephadex G-100 column (100 ×1.5 cm), packed andequiliberated with 0.05 M ammonium bicarbonate buffer.

Cloning and Expression of IL-9 Constructs in the pFLAG-1™ ExpressionVector

[0176] For the expression, purification and detection of human IL-9protein, IL-9 cDNA inserts were subcloned into the Xho2 site of themultiple cloning site (XhoI) of the 5.37 Kb Flag vector. FLAG technologyis centered on the fusion of a low molecular weight (1 kD), hydrophilic,FLAG marker peptide to the N-Terminus of a recombinant protein expressedby the pFLAG-1™ Expression Vector(1) (obtained from IBI Kodak). Eachbacterial colony was grown in LB broth containing 50 μg ampicillin perml until the optical density at 590 nm reached 0.6. IPTG was then addedto a final concentration of 1 mM, and the cultures incubated for anadditional 1 hr to-induce protein synthesis. The cells were harvested bycentrifugation, and the cell pellet was boiled in 50 μl of Laemmlibuffer (Laemmli, 1970) for 10 min and electrophoresed on 10%polyacrylamide gels. The Anti-FLAG™ M1 monoclonal antibody was used forspecific and efficient detection of the FLAG fusion protein on western(see FIG. 13) slot or dot blots throughout its expression, affinitypurification, and FLAG marker removal. The FLAG fusion protein wasrapidly purified under mild, non-denaturing conditions in a single stepby affinity chromatography with the murine Anti-FLAG™ IgG M1 monoclonalantibody covalently attached to agarose. Following affinity purificationthe fusion protein may be used after removal from the affinity column orthe authentic protein may be recovered in biologically active form byspecific and efficient proteolytic removal of the FLAG peptide withenterokinase. Final purification was achieved by chromatography onSephadex G-100 column (100×1.5 cm), packed and equiliberated with 0.05 Mammonium bicarbonate buffer. The promoters described above in thisexample may also be used with FLAG technology.

SDS-PAGE and Immunoblot Analysis

[0177] SDS-PAGE was performed by the method of Laemmli (Laemmli U.K.(1970) Nature 227, 680-685)(incorporated herein by reference in itsentirety) by using a 12.5% polyacrylamide gel in a mini-gel system (SE280 vertical gel unit, Hoefer). For immunoblot analysis, the proteinsseparated by SDS-PAGE were transferred to nitrocellulose membranes byusing the TE 22 Mighty small transfer unit (Hoefer) in 25 mMTris-glycine buffer, pH 8.3, containing 15% methanol (Towbin H., et al.,(1979) Proc. Natl. Acad. Sci. U.S.A. 76, 4350-4354). The unoccupiedbinding sites on the membrane were blocked by incubating for 1 h with 20mM Tris-HCl buffer, pH 8.0, containing 2% bovine serum albumin. Themembranes were then incubated with 1:200 dilution of antibodiesovernight at 4° C. The membranes were washed and treated with 1:2000diluted goat anti-rabbit IgG conjugated with either peroxidase oralkaline phosphotase for 1 h. After washing, the bound antibodies werevisualized by addition of the super-substrate chemiluminescent reagent(Pierce) or the 4-chloro-1-naphthol color developing reagent. Thereaction was stopped by immersing the membranes in distilled water. FIG.13 demonstrates that the purified recombinant human FLAG IL-9 fusionproteins (Met117 and Thr117) are the correct size and in the correctreading frame because they are recognized by the Anti-FLAG™ M1monoclonal antibody.

Analytical Methods

[0178] The molecular mass of the purified proteins was confirmed bymatrix assisted laser desorption mass spectrometry using PerceptiveBiosystems Voyager Biospectrometry workstation. Amino acid analyses wereperformed after hydrolysis of the sample in 6N HCl at 110-C for 24 h inevacuated sealed glass bulbs.

Automated Edman Degradation

[0179] The partial amino acid sequence of the purified proteins isdetermined by automated step-wise sequencing on an Applied Biosystemsmodel 477A gas-phase sequencer with an on-line model 20A PTH analyzer.

EXAMPLE 8 Deletion of Exon 2 and/or Exon 3: Mutagenesis and Sequencingof Constructs

[0180] Human exon 2 and exon 3 deletions are created using ExSitePCR-based site-directed mutagenesis kit as suggested by the manufacturer(Stratagene, La Jolla, Calif.). The PCR primers are as follows: h9CD1Uforward 5′-GTG ACC AGT TGT CTC TGT TTG-3′ (SEQ ID NO: 5); h9CD1L reverse5′-CTG CAT CTT GTT GAT GAG GAA-3′ (SEQ ID NO: 6); h9CD2U forward 5′-GACAAC TGC ACC AGA CCA TGC-3′ (SEQ ID NO: 7); h9CD2L reverse 5′-ATT AGC ACTGCA GTG GCA CTT-3′ (SEQ ID NO: 8). Exon 2 deletions are created by usingthe primer pair h9CD1L forward and h9CD1L reverse. Exon 3 deletions arecreated by using h9CD2U forward and h9CD2L reverse. Deletions thatincluded exon 2 and exon 3 use the primer pair h9CD2U forward h9CD1Lreverse.

[0181] Mouse exon 2 and exon 3 deletions are created using ExSitePCR-based site-directed mutagenesis kit as suggested by the manufacturer(Stratagene, La Jolla, Calif.). The PCR primers are as follows: m9CD1Uforward 5′-GTG ACC AGC TGC TTG TGT CTC-3′ (SEQ ID NO: 9); m9CD1L reverse5′-CTT CAG ATT TTC AAT AAG GTA-3′ (SEQ ID NO: 10); m9CD2U forward 5′-GATGAT TGT ACC ACA CCG TGC-3′ (SEQ ID NO: 11); m9CD2L reverse 5′-GTT GCCGCT GCA GCT ACA TTT-3′ (SEQ ID NO: 12). Exon 2 deletions are created byusing the primer pair m9CD1U forward and m9CD1L reverse. Exon 3deletions are created by using m9CD2U forward and m9CD2L reverse.Deletions that included exon 2 and exon 3 use the primer pair m9CD2Uforward m9CD1L reverse.

[0182] Mutagenized constructs of the hIL-9 and mIL-9 cDNA inserts aresequenced by the dideoxy-mediated chain termination method (Sanger etal. (1977) Proc. Natl. Acad. Sci. USA 74:5463) (incorporated herein byreference in its entirety), using the M13 (-20) forward primer(5′-GTAAAACGACGGCCAGT-3′) (SEQ ID NO: 18) and Sequenase™ (USB), withanalysis by gel electrophoresis (Sambrook, J. et al. (1989) Molecularcloning: a laboratory manual Cold Spring Harbor Laboratory Press, NewYork). Mutants that lack exon 2, exon 3, or both exon 2 and exon 3, andare without Taq polymerase-induced sequence errors can be used to createexpression vectors.

EXAMPLE 9 Cell Lines, Cellular Proliferation Assays and Inhibition ofIL-9 Activity

[0183] Cell lines were used to assess the function of peptides,aminosterols, tyrophostins, rhIL-9, rmIL-9, and recombinant mutant formsof these proteins as well as all other compounds that block IL-9function. A proliferative response was measured and compared to each ofthe other cytokines, variant or mutant forms of Il-9, or IL-9antagonists. In addition, compounds were tested for their ability toantagonize the baseline proliferative response. Once a baselineproliferative response was established for a cytokine a statisticallysignificant loss of response in assays repeated three times intriplicate was considered evidence for antagonism. A true antagonisticresponse was differentiated from cellular toxicity by directobservation, trypan blue staining (a technique well known to one ofnormal skill in the art), and loss of acid phosphatase activity.Specificity was assessed for the antagonist by evaluating whether theactivity was substantially expressed against other proliferative agentssuch as steel factor, interleukin 3, or interleukin 4.

[0184] The MO7e line is a human megakaryoblastic cell line, cultured inRPMI 1640 (GIBCO/BRL, Gaithersburg, Md.), 20% Fetal Bovine Serum(Hyclone) and 10 ng/ml IL-3 (R&D Systems, Minneapolis, Minn.). The MJline is a cytokine independent human lymphoblastoid cell line grown inRPMI 1640 (GIBCO/BRL) K562 is a human erthroleukemia cell line, culturedin RPMI 1640 (GIBCO/BRL) and 10% fetal bovine serum (Hyclone). C8166-45is a IL-9 receptor bearing line, cultured in RPMI 1640 GIBCO/BRL) and10% Fetal bovine serum (Hyclone). All the cell lines respond tocytokines including IL-9. The cell lines are fed and reseeded at 2×10⁵cells/ml every 72 hours.

[0185] The cells were centrifuged for 10 minutes at 2000 rpm andresuspended in RPMI 1640 with 0.5% Bovine Serum Albumin (GIBCO/BRL,Gaithersburg, Md.) and insulin-transferrin-selenium (ITS) cofactors(GIBCO/BRL, Gaithersburg, Md.). Cells were counted using a hemocytometerand diluted to a concentration of 1×10⁵ cells/ml and plated in a 96-wellmicrotiter plate. Each well contained 0.15 or 0.2 ml giving a finalconcentration of 2×10⁴ cells per well.

[0186] MO7e cells were stimulated with 50 ng/ml Stem Cell Factor (SCF)(R&D Systems, Minneapolis, Minn.) alone, 50 ng/ml SCF plus 50 ng/ml IL-3(R&D Systems, Minneapolis, Minn.), or 50 ng/ml SCF plus 50 ng/ml IL-9. Acontrol was included which contains cells and basal media only. Serialdilutions of test compounds (i.e, recombinant IL-9 proteins, peptides,small molecules) were added to each test condition in triplicate. The MJcell line was used as an independent control for nonspecificcytotoxicity. Cultures were incubated for 72-96 hours at 37° C. in 5%CO₂.

[0187] Cell proliferation was assayed using the Abacus CellProliferation Kit (Clontech, Palo Alto, Calif.) which determines theamount of intracellular acid phosphatase present as an indication ofcell number. The substrate p-nitrophenyl phosphate (pNPP) was convertedby acid phosphatase to p-nitrophenol which was measured as an indicatorof enzyme concentration. pNPP was added to each well and incubated at37° C. for one hour. IN sodium hydroxide was then added to stop theenzymatic reaction, and the amount of p-nitrophenol was quantified usinga Dynatech 2000 plate reader (Dynatech Laboratories, Chantilly, Va.) at410 nm wavelength. Standard curves that compare cell number with opticalabsorbance were used to determine the linear range of the assay. Assayresults were only used when absorbance measurements are within thelinear range of the assay.

[0188]FIG. 14 illustrates the amino acid sequence of three peptideantagonists of IL-9 function. Each peptide was incubated with MO7e cellsand inhibition of cellular growth induced by IL-9 was determined bycomparison with control conditions (no peptide)(see FIGS. 15-17). Therewas no evidence for cytotoxicity with any of the peptides. PeptidesKP-16 and KP-20 are predicted to lie within two anti-parallelalpha-helicies and define a critical IL-9 receptor binding domain forthe IL-9 ligand. The protein polymorphism at codon 117 lies within KP-23and KP-24 which also exhibited antagonistic properties, furtherdemonstrating the importance of this region surrounding the site ofgenetic variation.

[0189]FIG. 18 illustrates the effect of tyrophostins (obtained fromCalbiochem) on the IL-9 dependent growth of MO7e cell in vitro. Eachtyrophostin was incubated with MO7e cells and inhibition of cellulargrowth induced by IL-9 was determined by comparison with controlconditions (no treatment). There was no evidence for cytotoxicity withany of the treatments. Tyrophostins B46 and B56 provided the greatestinhibition suggesting a common structure activity relationship.

[0190]FIG. 19 illustrates the effect of aminosterols isolated from theshark liver as set forth in U.S. Ser. Nos. 08/290,826, 08/416,883,08/478,763, and/or 08/483,059, incorporated herein by reference on theIL-9 dependent growth of MO7e cell in vitro. Each aminosterol wasincubated with MO7e cells at 20 μg/ml of the culture media andinhibition of cellular growth induced by IL-9 was determined bycomparison with control conditions (no treatment). There was no evidencefor cytotoxicity with any of the treatments. Aminosterols 3 and 6consistently provided the greatest inhibition of growth.

EXAMPLE 10 Assay for Proliferation of IgE Secreting Cells

[0191] B cell lines can be used to assess the function of rIL-9 andrecombinant mutant forms of these proteins as well as other IL-9antagonists. The proliferation of IgE secreting cells is measured forrIL-9 and compared to other cytokines or variant forms of rIL-9. Inaddition, compounds are tested for their ability to antagonize thebaseline proliferative response of IgE secreting cells to rIL-9. Once abaseline IgE response is established for a cytokine, a statisticallysignificant (P<0.05) loss of response in assays repeated three times intriplicate is considered evidence for antagonism. A true antagonisticresponse is differentiated from cellular toxicity by trypan bluestaining (a technique well known to one of normal skill in the art).

Cell Preparation and Cultures

[0192] Peripheral blood lymphocytes (PBL) are isolated from heparinizedblood of healthy donors or by mincing the spleens of mice. Mononuclearcells are separated by centrifugation on Ficoll/Hypaque (Pharmacia,Uppsala, Sweden) gradients. Semi-purified human B lymphocytes areobtained by resetting with neuraminidase (Behring, Marburg, FRG)—treatedsheep red blood cells and plastic adherence for 1 hour at 37° C. B cellsare also purified using paramagnetic separation with anti-CD20 coatedmagnetic beads (DYNAL) according to the manufacturer's recommendations.

[0193] The relative proportion of B cells, T cells and monocytes isdetermined by flow cytometry using monoclonal antibodies specific forCD23, CD3 and CD14, respectively (Becton Dickinson, Mountain View,Calif.). Briefly, 106 cells/ml are incubated with a 1:1000 dilution ofphycoerythrin conjugated anti-CD23 and fluorescein-conjugated anti-CD3and anti-CD14 antibodies for 30 minutes at 4° C. After 3 spin washeswith sterile PBS and 1% bovine serum albumin (Sigma) fluorescence ismeasured with a cytofluorograph (FACSTAR Plus, Becton Dickinson,Grenoble, France). Typically, there are 45% CD20+, 35% CD3+ and 10%CD14+ cells in a count of 5000 cells per sample.

[0194] Cells are cultured at a density of 2×10⁶ cells/ml RPM1 1640supplemented with 10% heat-inactivated fetal calf serum (FCS), 2 mMglutamine, 100 U/ml penicillin, 100 μg/ml streptomycin and 20 mM HEPES(RPM1-FCS) at 37° C. under a 5% CO2/95% air humidified atmosphere.Cultures are incubated with increasing concentrations of IL-4, rhIL-9,rmIL-9, or recombinant mutant forms of these proteins, alone, or incombination. Competition experiments are run with mixtures of one ormore of these recombinant molecules or other IL-9 antagonists. Thecultures are maintained for 9-13 days.

Frequency of IgE-secreting B Cells

[0195] The frequency of IgE-secreting human B cells in response to humanor murine IL-9 is determined using an ELISA-spot assay (Dugas et al.,1993, Eur J Immunol 23:1687-1692; Renz, H. et al., 1990. J. Immunology.145:3641. Nitrocellulose flat-bottomed 96-well plates are coatedovernight at 4° C. with purified goat antihuman IgE mAb diluted in 0.1 MNaHCO3 buffer (2.5 μg/ml). After one PBS-Tween 20 wash, plates areincubated for 1 hour with RPMI-FCS to saturate nonspecific bindingsites. B cells obtained after 9-13 days of culture are collected, washedthrice and resuspended at 10⁵ cells/ml RPMI-FCS, then transferred ontothe anti-IgE-coated plates followed by an 18 hour incubation at 37° C. Aperoxidase-conjugated mouse antihuman IgE mab at various dilutions isadded for 2 hours at 37° C. after washing. Spots are visualized afteraddition of diamino-benzidine diluted in 0.1 M Tris-HCl containing 0.03%H₂O₂. After 24 hours spots are counted with an inverted microscope at25× magnification. Data are expressed as the number of IgE-secretingcells per 10⁶ cells.

EXAMPLE 11 ELISA for IgE Secreted by Cells Co-stimulated with IL-9

[0196] Cells are isolated, prepared and stimulated as described above inExample 10. Flat bottom microtiter plates (Nunc) are coated with rabbitanti-human IgE (1:2000, final dilution; Serotec, Oxford, GB), in 200 μlof 10 mM bicarbonate buffer (pH 9.6). After overnight incubation at 4°C., the plates are washed four times with phosphate-buffered saline(PBS) containing 0.05% Tween (PBS-Tween; Merck, Hohenbrunn, FRG) and areincubated for 1 h at room temperature with RPMI-FCS to saturatenonspecific protein-binding sites. After washing, 200 μl serialdilutions of human IgE (Eurobio, Les Ulis, France), standards inPBS-Tween are added to the respective plates to establish calibrationcurves. Dilutions of culture supernatants to be tested are then addedand, after 2 h at room temperature, the plates are washed and 200 μl ofdiluted specific alkaline phosphatase-conjugated anti-IgE (1:250;Serotec), anti-IgG or anti-IgM (Behring) is added in the appropriateplates. After 2 h at room temperature, the plates are washed and 200 μl(0.5 mg/ml) p-nitrophenylphosphate (Sigma) in citrate buffer is added.Plates are incubated at 37° C., and absorbance (A) is measured at 405 nmusing an autoreader (Dynatech Laboratories Inc, Chantilly, Va.). Thethreshold sensitivities of the assays are 100 pg/ml for IgE, 1 ng/ml forIgG, and 2 ng/ml for IgM and the variation between duplicatedeterminations of samples typically does not exceed 10%.

EXAMPLE 12 The Role of IL-9 in Murine Models of Asthma: The AirwayResponse of Unsensitized Animals Animals

[0197] Certified virus-free male mice ranging in age from 5 to 6 weekswere obtained from the Jackson Laboratory (Bar Harbor, Me.). Animalswere housed in high-efficiency particulate filtered air (HEPA) laminarflow hoods in a virus and antigen free facility and allowed free accessto pelleted rodent chow and water for 3 to 7 days prior to experimentalmanipulation. The animal facilities were maintained at 22° C. and thelight:dark cycle was automatically controlled (10:14 h light:dark). Maleand female DBA/2 (D2), C57BL/6 (B6), and (B6D2)F1 (F1) mice 5 to 6 weeksof age were purchased from the Jackson Laboratory, Bar Harbor, Me., orthe National Cancer Institute, Frederick, Md. BXD mice were purchasedfrom the Jackson Laboratory, Bar Harbor, Me. Food and water were presentad libitum.

Phenotyping and Efficacy of Pretreatment

[0198] To determine the bronchoconstrictor response, respiratory systempressure was measured at the trachea and recorded before and duringexposure to the drug. Mice were anesthetized and instrumented aspreviously described. (Levitt R C, and Mitzner W, FASEB J 2:2605-2608(1988); Levitt R C, and Mitzner W, J Appl Physiol 67(3): 1125-1132(1989); Kleeberger S, Bassett D, Jakab G J, and Levitt R C, Am J Physiol258(2)L313-320 (1990); Levitt R C; Pharmacogenetics 1:94-97 (1991);Levitt R C, and Ewart S L; Am J of Respir Crit Care bed151:1537-1542(1995); Ewart S, Levitt R C, and Mitzner W, In press, JAppl Phys (1995). Airway responsiveness was measured to one or more ofthe following: 5-hydroxytryptamine (5HT) (sigma). An additional branchconstruction that can be used is acetylcholine (sigma), atracurium(Glaxo welcome). A simple and repeatable measure of the change in P_(pi)following bronchoconstrictor challenge was used and which has beentermed the Airway Pressure Time Index (APTI) (Levitt R C, and Mitzner W,FASEB J 2:2605-2608 (1988); Levitt R C, and Mitzner W, J Appl Physiol67(3): 1125-1132 (1989). The APTI was assessed by the change in peakinspiratory pressure (Ppi) integrated from the time of injection tillthe peak pressure returned to baseline or plateaued. The APTI wascomparable to airway resistance (Rrs), however, the APTI includes anadditional component related to the recovery from bronchoconstriction.

[0199] The strain distribution of bronchial responsiveness wasidentified in multiple inbred mouse strains in previous studies (LevittR C, and Mitzner W, FASEB J 2:2605-2608 (1988); Levitt R C, and MitznerW, J Appl Physiol 67(3): 1125-1132 (1989). The Rrs and/or APTI wasdetermined in A/J, C3H/HeJ, DBA/2J, C57BL/6J mice.

[0200] Prior to sacrifice whole blood was collected for serum IgEmeasurements by needle puncture of the inferior vena cava in completelyanesthetized animals. The samples were spun to separate cells and serumwas collected and used to measure total IgE levels. Samples not measuredimmediately were frozen at −20° C.

[0201] Bronchoalveolar lavage and cellular analyses was preformed asdescribed elsewhere (Kleeberger et al., 1990).

[0202] All IgE serum samples were measured using an ELISAantibody-sandwich assay. Microtiter plates (Corning #2585096, Corning,N.Y.) were coated, 50 μl per well, with rat anti-mouse IgE antibody(Southern Biotechnology #1130-01, Birmingham, Ala.) at a concentrationof 2.5 μg/ml in coating buffer of sodium carbonate-sodium bicarbonatewith sodium azide (Sigma #S-7795, #S-6014 and #S-8032, St Louis, Mo.).Plates were covered with plastic wrap and incubated at 4° C. for 16hours. The plates were washed three times with a wash buffer of 0.05%Tween-20 (Sigma #P-7949) in phosphate-buffered saline (BioFluids #313,Rockville, Md.), incubating for five minutes for each wash. Blocking ofnonspecific binding sites was accomplished by adding 200 μl per well 5%bovine serum albumin (Sigma #A-7888) in PBS, covering with plastic wrapand incubating for 2 hours at 37° C. After washing three times with washbuffer, duplicate 50 Al test samples were added to the wells. Testsamples were assayed after being diluted 1:10, 1:50, and 1:100 with 5%BSA in wash buffer. In addition to the test samples a set of IgEstandards (PharMingen #03121D, San Diego, Calif.) at concentrations from0.8 ng/ml to 200 ng/ml in 5% BSA in wash buffer were assayed to generatea standard curve. A blank of no sample or standard was used to zero theplate reader (background). After adding samples and standards, the platewas covered with plastic wrap and incubated for 2 hours at roomtemperature. After washing three times with wash buffer, 50 ul of secondantibody rat anti-mouse IgE-horseradish peroxidase conjugate (PharMingen#02137E) was added at a concentration of 250 ng/ml in 5% BSA in washbuffer. The plate was covered with plastic wrap and incubated 2 hours atroom temperature. After washing three times with wash buffer, 100 ul ofthe substrate 0.5 mg/ml O-phenylaminediamine (Sigma #P-1526) in 0.1 Mcitrate buffer (Sigma #C-8532) was added to every well. After 5-10minutes the reaction was stopped with 50 μl of 12.5% H₂SO₄ (VWR #3370-4,Bridgeport, N.J.) and absorbance was measured at 490 nm on a DynatechMR-5000 plate reader (Chantilly, Va.). A standard curve was constructedfrom the standard IgE concentrations with antigen concentration on the xaxis (log scale) and absorbance on the y-axis (linear scale). Theconcentration of IgE in the samples was interpolated from the standardcurve.

EXAMPLE 13 The Role of IL-9 in Murine Models of Asthma: The AirwayResponse of Sensitized Animals Animals, Phenotyping, and Optimization ofAntigen Sensitization

[0203] Animals and handling were essentially as described in Example 12.Sensitization with turkey egg albumin (OVA) and aerosol challenge wascarried out to assess the effect on BHR, BAL, and serum IgE. OVA wasinjected I.P. (25 μg) day 0 prior to OVA or saline aerosolization. Micewere challenged with OVA or saline aerosolization which was given oncedaily for 5 to 7 days starting on either day 13 or 14. Phenotypicmeasurements of serum IgE, BAL, and BHR was carried out on day 21. Theeffect of a 7 day OVA aerosol exposure on bronchoconstrictor challengewith 5-HT and acetylcholine were evaluated along with serum total IgE,BAL total cell counts and differential cell counts, and bronchialresponsiveness. The effect of antibody (Ab) or saline pretreatment onsaline aerosol or OVA aerosol induced lung inflammation was examined bymeasuring BHR, BAL, and serum IgE. Ab were administered I.P. 2-3 daysprior to aerosolization of saline or OVA.

[0204] Lung histology was carried out after the lungs are removed duringdeep anesthesia. Since prior instrumentation may introduce artifact,separate animals were used for these studies. Thus, a small group ofanimals was treated in parallel exactly the same as the cohortundergoing various pretreatments except these animals were not used forother tests aside from bronchial responsiveness testing. After bronchialresponsiveness testing, the lungs were removed and submersed in liquidnitrogen. Cryosectioning and histologic examination were carried out ina routine fashion.

[0205] Polyclonal neutralizing antibodies for murine IL-9 were purchasedfrom R & D systems, Minneapolis, Minn. and blocking antibodies formurine IL-9 receptor were produced for Magainin Pharmaceuticals Inc. byLampine Biological Labatories, Ottsville, Pa. using peptide conjugatesproduced at Magainin. The polyclonal antisera were prepared in-rabbitsagainst peptide sequences from the murine IL-9 receptor. The peptidesused to produce the antisera were: GGQKAGAFTC (residues 1-10)(SEQ IDNO:19); LSNSIYRIDCHWSAPELGQESR (residues 11-32)(SEQ ID NO:20); andCESYEDKTEGEYYKSHWSEWS (residues 184-203 with a Cys residue added to theN-terminus for coupling the peptide to the carrier protein)(SEQ IDNO:21). The antisera were generated using techniques described inProtocols in Immunology, Chapter 9, Wiley. Briefly, the peptides werecoupled to the carrier protein, Keyhole Limpet, hemocyanin,(Sigma)through the side chain of the Cys residue using the bifunctionalcross-linking agent MBS(Pierce). Peptide conjugates were used toimmunize rabbits with appropriate adjuvents and useful antisera wasobtained after several booster injections of the peptide conjugate. Theantibodies were used therapeutically to down regulate the functions ofIL-9 and assess the importance of this pathway to baseline lungresponsiveness, serum IgE, and BAL in the unsensitized mouse. After Abpretreatment on baseline BHR, BAL, and serum IgE levels relative tocontrols was determined. In additional experiments, recombinant humanand murine IL-9 were administered I.P. 1 day before and daily duringantigen sensitization (days 13-18). The animals were then phenotyped asdescribed.

[0206] The phenotypic response of a representative animal treated withsaline I.P. on day zero and challenged on days 14-20 with saline (asdescribed in Example 12) is shown in FIG. 20 panel 1 (top). Baseline(control) serum total IgE was 9.2 ng/ml. Bronchoalveolar lavage (BAL)total cell counts showed 182,500 cells per milliliter of BAL. Theseanimals did not demonstrate bronchial hyperresponsiveness when comparedto historical controls (Levitt R C, and Mitzner W, J Appl Physiol 67(3):1125-1132;1989).

[0207]FIG. 20 panel 2 (top middle) shows a representative animal from agroup presensitized with OVA I.P on day zero and challenged with salineon days 14-20. These animals did not differ in their response tobronchoconstrictor, serum IgE, or BAL cell counts from the unsensitizedmice (FIG. 7 top panel).

[0208]FIG. 20 panel 3 (bottom middle) shows a representative animal fromthose presensitized with OVA I.P on day zero and challenged with antigen(OVA) on days 14-20. These animals developed bronchialhyperresponsiveness (approximately two to three-fold over controls),elevated serum IgE (nearly one thousand-fold over controls), andincreased numbers of inflammatory cells in the airway as demonstrated byelevated BAL cell counts (approximately thirty-fold) as compared tocontrols (FIG. 20 top 2 panels). Most of the cells recruited to theairway as a result of this antigen challenge were eosinophils.

[0209]FIG. 20 panel 4 (bottom) shows a representative animal from thosepresensitized with OVA I.P on day zero, pretreated with polyclonalneutralizing antibodies for murine IL-9 (approximately 200 μg/mouse I.P.in 0.5 ml of PBS), and challenged with antigen (OVA) on days 14-20.These animals were protected from the response to antigen. They did notdiffer significantly in their bronchial responsiveness, serum IgE, orBAL cell counts from controls (FIG. 20 top 2 panels).

[0210]FIG. 21 illustrates the effect of antigen challenge to OVA (asdescribed above) with and without pretreatment with polyclonalneutralizing antibodies to murine IL-9 I.P. three days prior inrepresentative animals. The left figure (A1-2-1B) is a histologicsection from the lungs of control animals (sensitized to OVA but exposedonly to a saline aerosol challenge). The middle figure (A1-3-5) is ahistologic section from the lungs of animals sensitized to OVA andexposed to an OVA aerosol challenge. The right figure (A1-4-5) is ahistologic section from the lungs of animals sensitized to OVA andexposed to an OVA aerosol challenge who were pretreated three days priorwith polyclonal neutralizing antibodies to murine IL-9. Pretreatmentwith neutralizing antibody produced histological confirmation ofcomplete protection from antigen challenge.

[0211]FIG. 22 panel 1 (top) shows a representative animal from micepresensitized with OVA I.P on day zero and challenged with antigen (OVA)on days 13-18. These animals developed bronchial hyperresponsiveness(approximately two to three-fold over controls), and increased numbersof inflammatory cells including eosinphils in the airways asdemonstrated by elevated BAL cell counts as compared to controls (FIG.20 top 2 panels). Many of the cells recruited to the airway as a resultof this antigen challenge were eosinophils.

[0212]FIG. 22 panel 2 (bottom) shows a representative animal from thosepresensitized with OVA I.P on day zero, pretreated with polyclonalneutralizing antibodies to the murine IL-9 receptor (approximately 1mg/mouse I.P. in 0.5 ml of PBS), and challenged with antigen (OVA) ondays 13-18. This representative animal was protected from the responseto antigen. This response did not differ significantly bronchialresponsiveness, BAL cell counts from controls (FIG. 20 top 2 panels).These data demonstrate the potential effectiveness of treating atopicallergy with antibodies to the IL-9 receptor.

EXAMPLE 14 Murine Spleen Isolation and Culture

[0213] Mice were anesthetized and spleens were removed aseptically.Spleens were minced with scissors and gently passed through a wire mesh(autoclaved) [#60 sieve]. Cells were resuspended in 40 mls of RPMI-1640[GIBCO, BRL, Rockville, Md.], and spun for 5 min. at 250×G twice. Thepellet was resuspended in 10 mls of lysing butter to remove RBCs [4.15gm NH4Cl, 0.5 gm KHCO3; 019 g EDTA to 500 mls with ddH20]. Cells wereincubated for about 5 minutes at 37° C. and 40 mls of RPMI-FCS[RPMI-1640, 10% AFBS, 50 μM BME 2 mM glutamine, containing penicillinand streptomycin]. These cells were spun again for 5 minutes at 250×Gand resuspended in 20 mls RPMI-FCS with or without 5 μg/ml ofconcanavalin A [Sigma #C5275]. IL-9 was assessed at 48 hours inuntreated splenocytes and after concanavalin A stimulation from DBA/2J(D2) and C57BL/6J (B6) mice. IL-9 was amplified by RT-PCR (as set forthin Example 6), and probed with an IL-9 specific murine probe afterSouthern transfer. Southern blots were performed by “standard”techniques. Briefly, RT-PCR products were electrophoresed in 2% agarosegels. Gels were stained with ethidium bromide and photographed with aruler to determine molecular weight of DNA in southern blot. Gels werethen soaked in 0.5N NaOH for 30 minutes and neutralized in 0.5M Tris, pH7.0 for 30 minutes. DNA was transferred to zetaprobe (BioRAD) nylonmembrane by capillary transfer in 20×SSC overnight. The next day, themembrane was air dried, baked at 80° C. for 15 minutes and prehybridizedin 6×SSC and 0.1% SDS for 1 hour at 42° C. A kinase end-labelled p32oligonucleotide probe (5′-AATTACCTTATTGAAAATCTGAAG-3′) was added to thehybridization solution plus 0.1 mg/ml sheared salmon sperm DNA andincubated overnight at 42° C. The next day, the filter was washed in3×SSC and 0.1 k SDS at 37° C. for 30 minutes, and the filter was exposedto film for 1 hour. FIG. 26 illustrates steady state levels of IL-9after 48 hours from each strain of mice. IL-9 was observed inunstimulated D2 (D2−) splenocytes, whereas no IL-9 was detectable in B6(B6−) mice. While there was a significant increase of IL-9 afterconcanavalin A stimulation in D2 (D2+) splenocytes, there was nodetectable It-9 in B6 (B6+) mice despite concanavalin A treatment.

EXAMPLE 15 Expression of Human Met117 IL-9 and Thr117 IL-9 in PBMCsSDS-PAGE and Immunoblot Analysis

[0214] After obtaining proteins isolated from human PBMC of healthydonors inhibiting either the wild type (Thr117) or Met117-IL-9 genotypesas set forth in Example 13, and SDS-PAGE was performed by the method ofLaemmli (Laemmli U.K. (1970) Nature 227, 680-685) by using a 18%polyacrylamide gel in a mini-gel system (Xcell II vertical gel unit,Novex). For immunoblot analysis, the proteins separated by SDS-PAGE weretransferred to nitrocellulose membranes by using the SD transblottransfer unit (Biorad) in 25 mM Tris-glycine buffer, pH 8.3, containing15% methanol (Towbin H., et al., (1979) (Proc. Natl. Acad. Sci. U.S.A.76, 4350-4354). The unoccupied binding cites on the membrane wereblocked by incubating for 1 hour to overnight with 20 mM Tris-HClbuffer, pH 8.0, plus 0.05% tween 20 (TBST) containing 5% dry milk. Themembranes were then incubated with 1:1000 dilution of goat anti-humanIL-9 polyclonal antibody (R&D Systems) for 1 hour at room temperature.The membranes were washed with TBST and treated with 1:10,000 dilutionof mouse anti-goat TgG conjugated with horseradish peroxidase for 1hour. After washing with TBST, the bound antibodies were visualized byaddition of the super signal substrate chemiluminescence system(Pierce).

[0215]FIG. 24 demonstrates the expression of human IL-9 proteins fromcultured PBMCs 48 hours after mitogen stimulation in individuals whosegenotypes have been determined by genomic analysis of the IL-9 gene.Lane 1 is molecular weight markers, lane 2 is a Met117 homozygote, lane3 is a heterozygote Met117/Thr117, lane four is a Thr117 homozygote. Asingle product of the approximate expected size (14 kD) was seen in eachindividual PBMCs after mitogen stimulation. These data demonstrate thatboth forms of the IL-9 protein are expressed and stable at steady state.

[0216] While the invention has been described and illustrated herein byreferences to various specific materials, procedures and examples, it isunderstood that the invention is not restricted to the particularmaterial combinations of material, and procedures selected for thatpurpose. Numerous variations of such details can be implied as will beappreciated by those skilled in the art.

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[0312] Other embodiments of the invention described above and will beapparent to those skilled in the art from consideration of thespecification and practice of the invention disclosed within. It isintended that the specification and examples considered as exemplaryonly, with true scope and spirit of the invention being indicated by thefollowing claims:

1 44 1 28 DNA Artificial sequence PCR oligonucleotide primer 1tctcgagcag gggtgtccaa ccttggcg 28 2 31 DNA Artificial sequence PCRoligonucleotide primer 2 gcagctggga taaataatat ttcatcttca t 31 3 31 DNAArtificial sequence PCR oligonucleotide primer 3 tctcgagcag agatgcagcaccacatgggg c 31 4 31 DNA Artificial sequence PCR oligonucleotide primer4 gcagctggta acagttatgg aggggaggtt t 31 5 21 DNA Artificial sequence PCRoligonucleotide primer 5 gtgaccagtt gtctctgttt g 21 6 21 DNA Artificialsequence PCR oligonucleotide primer 6 ctgcatcttg ttgatgagga a 21 7 21DNA Artificial sequence PCR oligonucleotide primer 7 gacaactgcaccagaccatg c 21 8 21 DNA Artificial sequence PCR oligonucleotide primer8 attagcactg cagtggcact t 21 9 21 DNA Artificial sequence PCRoligonucleotide primer 9 gtgaccagct gcttgtgtct c 21 10 21 DNA Artificialsequence PCR oligonucleotide primer 10 cttcagattt tcaataaggt a 21 11 21DNA Artificial sequence PCR oligonucleotide primer 11 gatgattgtaccacaccgtg c 21 12 21 DNA Artificial sequence PCR oligonucleotide primer12 gttgccgctg cagctacatt t 21 13 18 PRT Artificial sequence Peptidesequence 13 Ser Asp Asn Ala Thr Arg Pro Ala Phe Ser Glu Arg Leu Ser GlnMet 1 5 10 15 Thr Asn 14 18 PRT Artificial sequence Peptide sequence 14Phe Ser Arg Val Lys Lys Ser Val Glu Val Leu Lys Asn Asn Lys Ala 1 5 1015 Pro Tyr 15 18 PRT Artificial sequence Peptide sequence 15 Glu Gln ProAla Asn Gln Thr Thr Ala Gly Asn Ala Leu Thr Phe Leu 1 5 10 15 Lys Ser 1618 PRT Artificial sequence Residues 99-116 of Mature hIL-9 Receptor 16Thr Ala Gly Asn Ala Leu Thr Phe Leu Lys Ser Leu Leu Glu Ile Phe 1 5 1015 Gln Lys 17 17 DNA Artificial sequence Oligonucleotide primer 17gtaaaacgac ggccagt 17 18 17 DNA Artificial sequence Oligonucleotideprimer 18 gtaaaacgac ggccagt 17 19 10 PRT Mus musculus misc_featureMurine IL-9 Receptor Peptide Sequence 19 Gly Gly Gln Lys Ala Gly Ala PheThr Cys 1 5 10 20 22 PRT Mus musculus misc_feature Murine IL-9 ReceptorPeptide Sequence 20 Leu Ser Asn Ser Ile Tyr Arg Ile Asp Cys His Trp SerAla Pro Glu 1 5 10 15 Leu Gly Gln Glu Ser Arg 20 21 21 PRT Mus musculusmisc_feature Murine IL-9 Receptor Peptide Sequence 21 Cys Glu Ser TyrGlu Asp Lys Thr Glu Gly Glu Tyr Tyr Lys Ser His 1 5 10 15 Trp Ser GluTrp Ser 20 22 7 PRT Artificial sequence Residues 8-14 of Mature hIL-9Receptor 22 Thr Cys Leu Thr Asn Asn Ile 1 5 23 18 PRT Artificialsequence Residues 50-67 of Mature hIL-9 Receptor 23 Cys Phe Ser Glu ArgLeu Ser Gln Met Thr Asn Thr Thr Met Gln Thr 1 5 10 15 Arg Tyr 24 415 DNAHomo sapiens 24 tctcgagcag gggtgtccaa ccttggcggg gatcctggac atcaacttcctcatcaacaa 60 gatgcaggaa gatccagctt ccaagtgcca ctgcagtgct aatgtgaccagttgtctctg 120 tttgggcatt ccctctgaca actgcaccag accatgcttc agtgagagactgtctcagat 180 gaccaatacc accatgcaaa caagataccc actgattttc agtcgggtgaaaaaatcagt 240 tgaagtacta aagaacaaca agtgtccata tttttcctgt gaacagccatgcaaccaaac 300 cacggcaggc aacgcgctga catttctgaa gagtcttctg gaaattttccagaaagaaaa 360 gatgagaggg atgagaggca agatatgaag atgaaatatt atttatcccagctgc 415 25 127 PRT Homo sapiens 25 Gln Gly Cys Pro Thr Leu Ala Gly IleLeu Asp Ile Asn Phe Leu Ile 1 5 10 15 Asn Lys Met Gln Glu Asp Pro AlaSer Lys Cys His Cys Ser Ala Asn 20 25 30 Val Thr Ser Cys Leu Cys Leu GlyIle Pro Ser Asp Asn Cys Thr Arg 35 40 45 Pro Cys Phe Ser Glu Arg Leu SerGln Met Thr Asn Thr Thr Met Gln 50 55 60 Thr Arg Tyr Pro Leu Ile Phe SerArg Val Lys Lys Ser Val Glu Val 65 70 75 80 Leu Lys Asn Asn Lys Cys ProTyr Phe Phe Ser Cys Glu Gln Pro Cys 85 90 95 Asn Gln Thr Thr Ala Gly AsnAla Leu Thr Phe Leu Lys Ser Leu Leu 100 105 110 Glu Ile Phe Gln Lys GluLys Met Arg Gly Met Arg Gly Lys Ile 115 120 125 26 415 DNA Homo sapiens26 tctcgagcag gggtgtccaa ccttggcggg gatcctggac atcaacttcc tcatcaacaa 60gatgcaggaa gatccagctt ccaagtgcca ctgcagtgct aatgtgacca gttgtctctg 120tttgggcatt ccctctgaca actgcaccag accatgcttc agtgagagac tgtctcagat 180gaccaatacc accatgcaaa caagataccc actgattttc agtcgggtga aaaaatcagt 240tgaagtacta aagaacaaca agtgtccata tttttcctgt gaacagccat gcaaccaaac 300catggcaggc aacgcgctga catttctgaa gagtcttctg gaaattttcc agaaagaaaa 360gatgagaggg atgagaggca agatatgaag atgaaatatt atttatccca gctgc 415 27 126PRT Homo sapiens 27 Gln Gly Cys Pro Thr Leu Ala Gly Ile Leu Asp Ile AsnPhe Leu Ile 1 5 10 15 Asn Lys Met Gln Glu Asp Pro Ala Ser Lys Cys HisCys Ser Ala Asn 20 25 30 Val Thr Ser Cys Leu Cys Leu Gly Ile Pro Ser AspAsn Cys Thr Arg 35 40 45 Pro Cys Phe Ser Glu Arg Leu Ser Gln Met Thr AsnThr Thr Met Gln 50 55 60 Thr Arg Tyr Pro Leu Ile Phe Ser Arg Val Lys LysSer Val Glu Val 65 70 75 80 Leu Lys Asn Asn Lys Cys Pro Tyr Phe Ser CysGlu Gln Pro Cys Asn 85 90 95 Gln Thr Met Ala Gly Asn Ala Leu Thr Phe LeuLys Ser Leu Leu Glu 100 105 110 Ile Phe Gln Lys Glu Lys Met Arg Gly MetArg Gly Lys Ile 115 120 125 28 585 DNA Homo sapiens 28 atgaaaaagacagctatcgc gattgcagtg gcactggctg gtttcgctac cgttgcgcaa 60 gctgactacaaggacgacga tgacaagctt gaattctcta gagatatcgt cgacagatct 120 ctcgagcaggggtgtccaac cttggcgggg atcctggaca tcaacttcct catcaacaag 180 atgcaggaagatccagcttc caagtgccac tgcagtgcta atgtgaccag ttgtctctgt 240 ttgggcattccctctgacaa ctgcaccaga ccatgcttca gtgagagact gtctcagatg 300 accaataccaccatgcaaac aagataccca ctgattttca gtcgggtgaa aaaatcagtt 360 gaagtactaaagaacaacaa gtgtccatat ttttcctgtg aacagccatg caaccaaacc 420 acggcaggcaacgcgctgac atttctgaag agtcttctgg aaattttcca gaaagaaaag 480 atgagagggatgagaggcaa gatatgaaga tgaaatatta tttatcccag ctgccaacgg 540 tagcgaaaccagccagtgcc actgcaatcg cgatagctgt ctttt 585 29 168 PRT Homo sapiens 29Met Lys Lys Thr Ala Ile Ala Ile Ala Val Ala Leu Ala Gly Phe Ala 1 5 1015 Thr Val Ala Gln Ala Asp Tyr Lys Asp Asp Asp Asp Lys Leu Glu Phe 20 2530 Ser Arg Asp Ile Val Asp Arg Ser Leu Glu Gln Gly Cys Pro Thr Leu 35 4045 Ala Gly Ile Leu Asp Ile Asn Phe Leu Ile Asn Lys Met Gln Glu Asp 50 5560 Pro Ala Ser Lys Cys His Cys Ser Ala Asn Val Thr Ser Cys Leu Cys 65 7075 80 Leu Gly Ile Pro Ser Asp Asn Cys Thr Arg Pro Cys Phe Ser Glu Arg 8590 95 Leu Ser Gln Met Thr Asn Thr Thr Met Gln Thr Arg Tyr Pro Leu Ile100 105 110 Phe Ser Arg Val Lys Lys Ser Val Glu Val Leu Lys Asn Asn LysCys 115 120 125 Pro Tyr Phe Ser Cys Glu Gln Pro Cys Asn Gln Thr Thr AlaGly Asn 130 135 140 Ala Leu Thr Phe Leu Lys Ser Leu Leu Glu Ile Phe GlnLys Glu Lys 145 150 155 160 Met Arg Gly Met Arg Gly Lys Ile 165 30 585DNA Homo sapiens 30 atgaaaaaga cagctatcgc gattgcagtg gcactggctggtttcgctac cgttgcgcaa 60 gctgactaca aggacgacga tgacaagctt gaattctctagagatatcgt cgacagatct 120 ctcgagcagg ggtgtccaac cttggcgggg atcctggacatcaacttcct catcaacaag 180 atgcaggaag atccagcttc caagtgccac tgcagtgctaatgtgaccag ttgtctctgt 240 ttgggcattc cctctgacaa ctgcaccaga ccatgcttcagtgagagact gtctcagatg 300 accaatacca ccatgcaaac aagataccca ctgattttcagtcgggtgaa aaaatcagtt 360 gaagtactaa agaacaacaa gtgtccatat ttttcctgtgaacagccatg caaccaaacc 420 atggcaggca acgcgctgac atttctgaag agtcttctggaaattttcca gaaagaaaag 480 atgagaggga tgagaggcaa gatatgaaga tgaaatattatttatcccag ctgccaacgg 540 tagcgaaacc agccagtgcc actgcaatcg cgatagctgtctttt 585 31 168 PRT Homo sapiens 31 Met Lys Lys Thr Ala Ile Ala Ile AlaVal Ala Leu Ala Gly Phe Ala 1 5 10 15 Thr Val Ala Gln Ala Asp Tyr LysAsp Asp Asp Asp Lys Leu Glu Phe 20 25 30 Ser Arg Asp Ile Val Asp Arg SerLeu Glu Gln Gly Cys Pro Thr Leu 35 40 45 Ala Gly Ile Leu Asp Ile Asn PheLeu Ile Asn Lys Met Gln Glu Asp 50 55 60 Pro Ala Ser Lys Cys His Cys SerAla Asn Val Thr Ser Cys Leu Cys 65 70 75 80 Leu Gly Ile Pro Ser Asp AsnCys Thr Arg Pro Cys Phe Ser Glu Arg 85 90 95 Leu Ser Gln Met Thr Asn ThrThr Met Gln Thr Arg Tyr Pro Leu Ile 100 105 110 Phe Ser Arg Val Lys LysSer Val Glu Val Leu Lys Asn Asn Lys Cys 115 120 125 Pro Tyr Phe Ser CysGlu Gln Pro Cys Asn Gln Thr Met Ala Gly Asn 130 135 140 Ala Leu Thr PheLeu Lys Ser Leu Leu Glu Ile Phe Gln Lys Glu Lys 145 150 155 160 Met ArgGly Met Arg Gly Lys Ile 165 32 18 DNA Artificial sequence PCRoligonucleotide primer 32 gctccagtcc gctgtcaa 18 33 18 DNA Artificialsequence PCR oligonucleotide primer 33 ctccccctgc agcctacc 18 34 20 DNAArtificial sequence PCR oligonucleotide primer 34 cggggctgac taaaggttct20 35 20 DNA Artificial sequence PCR oligonucleotide primer 35gttcttaaag agcattcact 20 36 24 DNA Artificial sequence PCRoligonucleotide primer 36 attttcacat ctggaatctt cact 24 37 20 DNAArtificial sequence PCR oligonucleotide primer 37 aatccaaggt caacattatg20 38 21 DNA Artificial sequence PCR oligonucleotide primer 38tttctttgaa taaatcctta c 21 39 21 DNA Artificial sequence PCRoligonucleotide primer 39 gaaatcacca acaggaacat a 21 40 21 DNAArtificial sequence PCR oligonucleotide primer 40 atcaactttc atccccacagt 21 41 24 DNA Artificial sequence PCR oligonucleotide primer 41ggataaataa tatttcatct tcat 24 42 21 DNA Artificial sequenceOligonucleotide primer 42 caaatctgaa gagcaaacta t 21 43 24 DNAArtificial sequence Oligonucleotide primer 43 ttaaaaaatt catttcagta ttct24 44 24 DNA Artificial sequence Kinase end-labeled p32 oligonucleotideprobe 44 aattacctta ttgaaaatct gaag 24

What is claimed is:
 1. A purified and isolated DNA molecule having anucleotide sequence encoding human interleukin-9 containing methionineat position 117 or fragments thereof.
 2. The purified and isolated DNAmolecule of claim 1 and degenerate sequences thereof.
 3. The purifiedand isolated DNA molecule of claim 1, wherein said DNA molecule isgenomic.
 4. The purified and isolated DNA molecule of claim 1, whereinsaid DNA molecule is used for the expression of IL-9 RNA.
 5. Thepurified and isolated DNA molecule of claim 1, wherein said DNA moleculeis used for the expression of IL-9 protein.
 6. A purified and isolatedprotein molecule selected from the group consisting of an amino acidsequence of human interleukin-9 containing methionine at position 117,fragments thereof, and sequences substantially homologous to an aminoacid sequence of human interleukin-9 containing methionine at position117.
 7. A chemically synthesized molecule selected from the groupconsisting of an amino acid sequence of human interleukin-9 containingmethionine at position 117, fragments thereof, and sequencessubstantially homologous to an amino acid sequence of humaninterleukin-9 containing methionine at position
 117. 8. A recombinantDNA molecule having a nucleotide sequence encoding human interleukin-9containing methionine at position 117 or a fragment thereof.
 9. Apurified and isolated RNA molecule having a nucleotide sequence encodinghuman interleukin-9 containing methionine at position 117 or fragmentsthereof.
 10. A method of alleviating asthma-related disorders bydown-regulating the activity of interleukin-9 by administering topatients in need of such treatment an effective amount of the purifiedand isolated DNA molecule of claim
 1. 11. A method of alleviatingasthma-related disorders by down-regulating the activity ofinterleukin-9 by administering to patients in need of such treatment aneffective amount of the purified and isolated RNA molecule of claim 9.12. A method of alleviating asthma-related disorders by down-regulatingthe activity of interleukin-9 by administering to patients in need ofsuch treatment an effective amount of the purified and isolated DNAmolecule of claim
 4. 13. A method of alleviating asthma-relateddisorders by down-regulating the activity of interleukin-9 byadministering to patients in need of such treatment an effective amountof the purified and isolated protein molecules of claim
 6. 14. A methodof alleviating asthma-related disorders by down-regulating the activityof interleukin-9 by administering to patients in need of such treatmentan effective amount of the chemically synthesized molecules of claim 7.15. A method of alleviating asthma-related disorders by down-regulatingthe activity of interleukin-9 by administering to patients in need ofsuch treatment an effective amount of the recombinant DNA molecule ofclaim
 8. 16. The purified and isolated protein molecule of claim 6,wherein said fragment of human interleukin-9 containing methionine atposition 117 having from about 3 to about 25 amino acids.
 17. Thechemically synthesized molecule of claim 7, wherein said fragment ofhuman interleukin-9 containing methionine at position 117 having fromabout 3 to about 25 amino acids.
 18. A method of alleviatingasthma-related disorders by down-regulating the activity ofinterleukin-9 by administering to patients in need of such treatment aneffective amount of a soluble interleukin-9 receptor or an activefragment thereof.
 19. A method of alleviating asthma-related disordersby down-regulating the activity of interleukin-9 by administering topatients in need of such treatment an effective amount of an antibodyspecific for human interleukin-9 or the interleukin-9 receptor that canbe administered in an amount sufficient to down regulate the activity ofinterleukin-9.
 20. An antibody specific for human interleukin-9, whereinsuch antibody can be administered in an amount sufficient to downregulate the activity of interleukin-9.
 21. An antibody specific for theinterleukin-9 receptor, wherein such antibody can be administered in anamount sufficient to down regulate the activity of interleukin-9. 22.Antisense DNA comprising the antisense sequence of human interleukin-9or active fragments thereof.
 23. A human interleukin-9 variant whereinthe threonine residue at position 117 of the native-sequence IL-9 isreplaced with another amino acid.
 24. The human interleukin-9 variantaccording to claim 23, wherein said threonine residue is replaced with ahydrophobic amino acid selected from the group consisting of Alanine,Valine, Leucine, Isoleucine, Proline, Methionine, Phenylalanine, andTryptophan.
 25. The human interleukin-9 variant according to claim 23,wherein said threonine residue is replaced with Methionine.
 26. Apurified and isolated molecule selected from a group of DNA and RNAhaving a nucleotide sequence encoding human interleukin-9 containing atleast 1 exon.
 27. The purified and isolated molecule of claim 26containing from about 1 to 5 exons.
 28. A purified and isolated moleculeof claim 26 having from about 1 to about 4 exons.
 29. A purified andisolated molecule of claim 26 having from about 1 to about 3 exons. 30.A purified and isolated molecule of claim 26 having from about 1 toabout 2 exons.
 31. The purified and isolated molecule of claim 26,wherein exon 2 is deleted.
 32. The purified and isolated molecule ofclaim 26, wherein exon 3 is deleted.
 33. The purified and isolatedmolecule of claim 26, wherein exons 2 and 3 are deleted.
 34. A method ofalleviating asthma-related disorders by down-regulating the activity ofinterleukin-9 by administering to patients in need of such treatment aneffective amount of the purified and isolated molecule of claim
 26. 35.A purified and isolated protein molecule having an amino acid sequenceencoding human interleukin-9 containing at least 1 exon.
 36. Thepurified and isolated protein molecule of claim 35 containing from about1 to 5 exons.
 37. The purified and isolated protein molecule of claim 35having from about 1 to about 4 exons.
 38. The purified and isolatedprotein molecule of claim 35 having from about 1 to about 3 exons. 39.The purified and isolated protein molecule of claim 35 having from about1 to about 2 exons.
 40. The purified and isolated protein molecule ofclaim 35, wherein exon 2 is deleted.
 41. The purified and isolatedprotein molecule of claim 35, wherein exon 3 is deleted.
 42. Thepurified and isolated protein molecule of claim 35, wherein exons 2 and3 are deleted.
 43. A method of alleviating asthma-related disorders bydown-regulating the activity of interleukin-9 by administering topatients in need of such treatment an effective amount of the purifiedand isolated protein molecule of claim
 35. 44. A chemically synthesizedmolecule of claim 7 further comprising one or more chemical moietiesattached thereto.
 45. A detectably labeled nucleic acid moleculehybridizable to the DNA molecule of claim
 1. 46. An oligonucleotideprimer for amplifying human DNA encoding an interleukin-9 molecule. 47.The oligonucleotide primer of claim 46, wherein the human DNA encoded isgenomic.
 48. The oligonucleotide primer of claim 46 encoding methionineat position 117 of the IL-9 or a fragment thereof.
 49. Anoligonucleotide primer having a sequence suitable for the amplificationof a IL-9 molecule comprising a methionine at position 117 of IL-9 or afragment thereof.
 50. An expression vector having a sequence of the DNAmolecule of claim 1 under control of regulatory elements permittingexpression of said sequence in a cell.
 51. A unicellular hosttransformed or transfected with the DNA molecule of claim
 1. 52. Theunicellular host of claim 51, wherein the unicellular host is selectedfrom the group consisting of bacteria, yeast, mammalian cells, plantcells, insect cells, and human cell in tissue culture.
 53. Theunicellular host of claim 51, wherein the unicellular host is selectedfrom the group consisting of E. coli, Pseudomonas, Bacillus,Streptomyces, yeast, CHO, R1.1, B-W, LM, COS 1, COS 7, BSC1, BSC40,BMT10, and Sf9 cells.
 54. The unicellular host of claim 51, wherein theunicellular host is a yeast selected from the group consisting ofSaccharomyces, Pichia, Candida, Hansenula, and Torulopis.
 55. Amammalian cell comprising a DNA sequence encoding an interleukin-9polypeptide and modified in vitro to permit enhanced expression of theinterleukin-9 polypeptide by a homologous recombinational eventcomprising the step of inserting an expression regulatory sequence infunctional proximity to the interleukin-9 polypeptide encoding sequence.56. The mammalian cell of claim 55, wherein said expression regulatorysequence in functional proximity to the interleukin-9 polypeptideencoding sequence is an interleukin-9 expression regulatory sequence andthe homologous recombinational event replaces a mutant interleukin-9polypeptide expression regulatory sequence.
 57. The mammalian cell ofclaim 55, wherein said expression regulatory sequence in functionalproximity to the interleukin-9 polypeptide encoding sequence is not aninterleukin-9 expression regulatory sequence.
 58. A method for preparingan interleukin-9 polypeptide comprising the steps of: (a) culturing theDNA molecule of claim 1 under conditions that provide for expression ofthe interleukin-9 polypeptide; and (b) recovering the expressedinterleukin-9 polypeptide.
 59. The antibody of claim 20 or 21, whereinsaid antibody is a monoclonal antibody.
 60. The antibody of claim 20 or21, wherein said antibody is labeled with a detectable label.
 61. A cellline that produces said monoclonal antibody of claim
 59. 62. A method ofpreparing an antibody specific to an interleukin-9 polypeptide whichcomprises the DNA molecule of claim 1 comprising the steps of: (a)conjugating an interleukin-9 polypeptide which corresponds to the DNAmolecule of claim 1 to a carrier protein; (b) immunizing a host animalwith the interleukin-9 polypeptide fragment-carrier protein conjugate ofstep (a) admixed with an adjuvant; and (c) obtaining antibody from theimmunized host animal.
 63. A method of quantifying interleukin-9polypeptide which comprises the DNA molecule of claim 1 comprising thesteps of: (a) contacting a sample suspected of containing interleukin-9polypeptides with an antibody that specifically binds to theinterleukin-9 polypeptides under conditions that allow for the formationof reaction complexes comprising the antibody and these interleukin-9polypeptides; and (b) detecting the formation of reaction complexescomprising the antibody and interleukin-9 polypeptides in the sample,wherein detection of the formation of reaction complexes indicates thepresence of interleukin-9 polypeptides in the sample.
 64. The method ofclaim 63, wherein said antibody is bound to a solid phase support. 65.An in vitro method for evaluating the levels of interleukin-9polypeptides containing methionine at position 117 or fragments thereofin a biologic sample comprising the steps of: (a) detecting theformation of reaction complexes in a biological sample according to themethod of claim 63, and (b) evaluating the amount of reaction complexesformed, which amount of reaction complexes corresponds to the level ofinterleukin-9 polypeptides in the biological sample.
 66. An in vitromethod for detecting or diagnosing susceptibility to atopy, asthma, or arelated disorder associated with elevated levels of interleukin-9polypeptide in a human subject comprising the steps of: (a) evaluatingthe level of interleukin-9 polypeptides in a biological sample; and (b)comparing the level of interleukin-9 polypeptides present in normalsubjects or in the subjects at an earlier time, wherein an increase inthe level of interleukin-9 polypeptides as compared to normal levelsindicates a predisposition to atopy, asthma, and related disorders. 67.The in vitro method of claim 66, wherein the interleukin-9 polypeptideevaluated is Theorine
 117. 68. An in vitro method for detecting ordiagnosing the presence of atopy, asthma, or related disordersassociated with a lack of a methionine at position 117 of theinterleukin-9 polypeptide in a human subject comprising: (a) evaluatingthe amount of a specific interleukin-9 polypeptide in a biologicalsample; and (b) comparing the level of interleukin-9 polypeptide presentin non-atopic subjects or in the subjects at an earlier time, wherein anincrease in the level of a specific interleukin-9 polypeptide ascompared to non-atopic levels indicates a predisposition to atopy,asthma, and related disorders.
 69. The in vitro method of claim 68,wherein the specific interleukin-9 polypeptide evaluated is Theorine117.
 70. An in vitro method for monitoring a therapeutic treatment ofatopy, asthma, and related disorders in a mammalian subject comprisingevaluating the levels of a specific interleukin-9 polypeptide in aseries of biologic samples obtained at different time points from ahuman subject undergoing therapeutic treatment with polypeptides havingthe sequence of human interleukin-9 containing methionine at position117 or fragments thereof.
 71. A pharmaceutical composition comprisingthe DNA molecule of claim 1 in a pharmaceutically acceptable carrier.72. A pharmaceutical composition comprising the protein molecule ofclaim 6 in a pharmaceutically acceptable carrier.
 73. A pharmaceuticalcomposition comprising the chemically synthesized molecule of claim 7 ina pharmaceutically acceptable carrier.
 74. A pharmaceutical compositioncomprising said antibody of claim 59 in a pharmaceutically acceptablecarrier.
 75. A pharmaceutical composition for decreasing the function ofinterleukin-9 polypeptide comprising an antagonist of interleukin-9polypeptide which comprises the chemically synthesized molecule of claim7 in a pharmaceutically acceptable carrier.
 76. A pharmaceuticalcomposition for decreasing the function of interleukin-9 polypeptidecomprising an antagonist of interleukin-9 polypeptide which comprisesthe protein molecule of claim 6 in a pharmaceutically acceptablecarrier.
 77. A pharmaceutical composition for decreasing the function ofinterleukin-9 polypeptide comprising an antagonist of interleukin-9polypeptide which comprises the DNA molecule of claim 1 in apharmaceutically acceptable carrier.
 78. The pharmaceutical compositionof claim 75, wherein the antagonist is selected from the groupcomprising an antibody that binds to the interleukin-9 polypeptide andan antibody that binds to the interleukin-9 receptor.
 79. Thepharmaceutical composition of claim 76, wherein the antagonist isselected from the group comprising an antibody that binds to theinterleukin-9 polypeptide and an antibody that binds to theinterleukin-9 receptor.
 80. The pharmaceutical composition of claim 77,wherein the antagonist is selected from the group comprising an antibodythat binds to the interleukin-9 polypeptide and an antibody that bindsto the interleukin-9 receptor.
 81. A method of alleviatingasthma-related disorders by administering to patents in need of such atreatment a compound that will down regulate the function of either IL-9or the IL-9 receptor.
 82. The method of claim 81, wherein said compoundis an inhibitor of the signal transduction of protein tyrosine kinase.83. The method of claim 82, wherein said inhibitor is selected fromkinase inhibitors comprising Tyrphostins.
 84. The method of claim 81,wherein said compound is selected from a group of aminosterols describedin FIG.
 28. 85. The method of claim 81, wherein the compound inhibitsthe interaction of IL-9 with the IL-9 receptor.
 86. The method of claim81, wherein the compound is a substitution or deletion analogue orfragment of human IL-9.
 87. The method of claim 86, wherein the compoundis sequence ID NO: 15(KP-23).
 88. The method of claim 86, wherein thecompound is sequence ID No: 16(KP-24).
 89. The method of claim 85,wherein the compound is human IL-9 containing a Met residue at position117 or a fragment thereof.
 90. The method of claim 89, wherein thecompound is sequence ID NO: 13(KP-16).
 91. The method of claim 89,wherein the compound is sequence ID NO: 14(KP-20).
 92. A cell having thepurified and isolated molecule of claim
 27. 93. A cell having thepurified and isolated molecule of claim
 28. 94. A cell having thepurified and isolated molecule of claim
 29. 95. A cell having thepurified and isolated molecule of claim
 30. 96. A cell having thepurified and isolated molecule of claim
 31. 97. A cell having thepurified and isolated molecule of claim
 32. 98. A cell having thepurified and isolated molecule of claim
 33. 99. The cell line of claim61 wherein the cell line is immortal.
 100. A composition comprising anIL-9 antagonist selected from the group consisting of peptides havingthe sequenceSer-Asp-Asn-Ala-Thr-Arg-Pro-Ala-Phe-Ser-Glu-Arg-Leu-Ser-Gln-Met-Thr-Asn(Seq. ID No. 13);Phe-Ser-Arg-Val-Lys-Lys-Ser-Val-Glu-Val-Leu-Lys-Asn-Asn-Lys-Ala-Pro-Tyr(Seq. ID No. 14); andGlu-Gln-Pro-Ala-Asn-Gln-Thr-Thr-Ala-Gly-Asn-Ala-Leu-Thr-Phe-Leu-Lys-Ser(Seq. ID No. 15).
 101. The method of claim 81 wherein the compound has aconfiguration substantially similar to the 3-D structure correspondingto amino acids 44-89 of human IL-9 or a fragment thereof.
 102. A segmentof human IL-9 that elicits antibodies that block the binding of humanIL-9 to its receptor.
 103. The IL-9 segment of claim 102 having asequence selected from the group consisting ofCys-Phe-Ser-Glu-Arg-Leu-Ser-Gln-Met-Thr-Asn-Thr-Thr-Met-Gln-Thr-Arg-Tyr(Seq. ID No. 23) andThr-Ala-Gly-Asn-Ala-Leu-Thr-Phe-Leu-Lys-Ser-Leu-Leu-Glu-Ile-Phe-Gln-Lys(Seq. ID No. 16).
 104. A segment of the human IL-9 receptor that elicitsantibodies that block the binding of human IL-9 to the receptor. 105.The human IL-9 receptor segment of claim 104 having the sequenceThr-Cys-Leu-Thr-Asn-Asn-Ile (Seq. ID No. 22).
 106. An epitope from humanIL-9 that binds to compounds that block the binding of IL-9 to itsreceptor.
 107. The epitope of claim 106 wherein the compound is anantibody.
 108. A method for identifying antagonists of IL-9 or the IL-9receptor comprising the steps of: a) obtaining animals susceptible toairway hyperresponsiveness; b) administering antigens that induce airwayhyperresponsivness; c) comparing the characteristics of any resultingairway hyperresponsiveness with the characteristics of airwayhyperresponsiveness obtained with pretreatment with a possible IL-9 orIL-9 receptor antagonist agent; and d) selecting those agents for whichpretreatment diminished the characteristics.
 109. The method of claim 9wherein the animal expresses the human IL-9 receptor.